bluuebunny/crossref_metadata_2025 huggingface dataset:
Perspective on the role of P2X‐purinoceptor activation in human vas deferens contractility
Authors: Amobi Nnaemeka I. B. , Guillebaud John , Smith I. Christopher H.
Published: January 2012

The contractile actions of α,β‐methylene ATP (α,β‐meATP) and ATP and the effects of K+ channel blockers in longitudinal and circular muscles of human vas deferens were investigated with a view to clarifying the functional importance of P2X1‐purinoceptor activation and K+ channels in modulating contractility of the tissues. The results provide an experiment‐based perspective for resolving differing reports on purinergic activation of the tissues and uncertain roles of large‐conductance Ca2+‐activated K+ (BKCa) and voltage‐gated delayed rectifier K+ (KV) channels. α,β‐Methylene ATP (3–100 μm) evoked suramin‐sensitive contractions of longitudinal muscle but rarely of circular muscle. ATP (0.1–3 mm) less reliably activated only longitudinal muscle contractions. These were enhanced by ARL 67156 (100 μm), but a different ectonucleotidase inhibitor, POM 1, was ineffective. Both muscle types were unresponsive to ADP‐βS (100 μm), a P2Y‐purinoceptor agonist. Longitudinal muscle contractions in response to α,β‐meATP were enhanced by FPL 64176 (1 μm), an L‐type Ca2+ agonist, TEA (1 mm), a non‐specific K+ channel blocker, 4‐aminopyridine (0.3 mm), a selective blocker of KV channels, and iberiotoxin (0.1 μm), a selective blocker of BKCa channels. Quiescent circular muscles responded to α,β‐meATP reliably in the presence of FPL 64176 or iberiotoxin. Apamin (0.1 μm), a selective blocker of small conductance Ca2+‐activated K+ (SKCa) channels had no effect in both muscle types. Y‐27632 (1–10 μm) reduced longitudinal muscle contractions in response to α,β‐meATP, suggesting involvement of Rho‐kinase‐dependent contractile mechanisms. The results indicate that P2X1‐purinoceptor stimulation elicits excitatory effects that: (a) lead to longitudinal muscle contraction and secondary activation of 4‐aminopyridine‐sensitive (KV) and iberiotoxin‐sensitive (BKCa) K+ channels; and (b) are subcontractile in circular muscle due to ancillary activation of BKCa channels. The novel finding of differential action by P2X1‐purinoceptor agonists in the muscle types has functional implication in terms of the purinergic contribution to overall contractile function of human vas deferens. The modulatory effects of KV and BKCa channels following P2X1‐purinoceptor activation may be pivotal in providing the crucial physiological mechanism that ensures temporal co‐ordination of longitudinal and circular muscle contractility.
Temperature‐dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion
Authors: Kalsi Kameljit K. , González‐Alonso José
Published: January 2012

Human limb muscle and skin blood flow increases significantly with elevations in temperature, possibly through physiological processes that involve temperature‐sensitive regulatory mechanisms. Here we tested the hypothesis that the release of the vasodilator ATP from human erythrocytes is sensitive to physiological increases in temperature both in vitro and in vivo, and examined potential channel/transporters involved. To investigate the source of ATP release, whole blood, red blood cells (RBCs), plasma and serum were heated in vitro to 33, 36, 39 and 42°C. In vitro heating augmented plasma or ‘bathing solution’ ATP in whole blood and RBC samples, but not in either isolated plasma or serum samples. Heat‐induced ATP release was blocked by niflumic acid and glibenclamide, but was not affected by inhibitors of nucleoside transport or anion exchange. Heating blood to 42°C enhanced (P < 0.05) membrane protein abundance of cystic fibrosis transmembrane conductance regulator (CFTR) in RBCs. In a parallel in vivo study in humans exposed to whole‐body heating at rest and during exercise, increases in muscle temperature from 35 to 40°C correlated strongly with elevations in arterial plasma ATP (r2 = 0.91; P = 0.0001), but not with femoral venous plasma ATP (r2 = 0.61; P = 0.14). In vitro, however, the increase in ATP release from RBCs was similar in arterial and venous samples heated to 39°C. Our findings demonstrate that erythrocyte ATP release is sensitive to physiological increases in temperature, possibly via activation of CFTR‐like channels, and suggest that temperature‐dependent release of ATP from erythrocytes might be an important mechanism regulating human limb muscle and skin perfusion in conditions that alter blood and tissue temperature.
Atomic layer deposition of Ru onto organic monolayers: Shifting metal effective work function using monolayer structure
Authors: Park Kie Jin , Parsons Gregory N.
Published: January 2012

Organic thin films are integrated with metal atomic layer deposition metallization to adjust and tune the electronic properties of metal/dielectric junctions. Specifically, 3-aminopropyltriethoxysilane (APTES) and undecenyltrichlorosilane (UDTS) self-assembled monolayers on hafnia-based dielectrics produce a shift in the effective work function of ruthenium metal deposited by atomic layer deposition at 325 °C onto the monolayer/oxide heterostructure. Based on infrared analysis, the molecular monolayer is relatively stable upon heating above 300 °C. The effective work function extracted from measurements of flat band voltage versus oxide thickness shift by +0.1 and −0.6 V when the metal is deposited on APTES and UDTS, respectively, consistent with expected organic interface dipoles on the dielectric surface. Results suggest various nucleation mechanisms for ALD growth on organic monolayers, and demonstrate that low temperature ALD is useful for metallization of organic modified surfaces for a range of applications.
Mechanisms for hydrophilic/hydrophobic wetting transitions on cellulose cotton fibers coated using Al2O3 atomic layer deposition
Authors: Lee Kyoungmi , Jur Jesse S. , Kim Do Han , Parsons Gregory N.
Published: January 2012

This report explores reactions that proceed during the first few cycles of inorganic film atomic layer deposition (ALD) on natural cellulose cotton fibers, and how surface reactions can explain the previously observed transitions in surface wetting upon ALD on cotton fibers. Atomic layer deposition of aluminum oxide and zinc oxide onto natural cotton cellulose produces a transition from hydrophilic to hydrophobic, then from hydrophobic back to hydrophilic, and we describe here the main factors that bring about. Interestingly, we show that air exposure and related adventitious carbon adsorption also affects the subsequent reactions and wetting properties obtained after subsequent ALD cycles. X-ray photoelectron spectroscopy and in situ Fourier transform infrared spectroscopy data indicate Al-(O-C-)3 bonding units form when trimethylaluminum interacts with surface –OH units during the first precursor doses, producing a hydrophobic finish on the cotton that remains for only a few ALD cycles. Also, field-emission scanning electron microscopy results show that some surface roughening may occur in the first few ALD cycles, and the roughening of the hydrophobic-finished surface can also promote an increase in measured hydrophobicity.
New Betaproteobacterial Rhizobium Strains Able To Efficiently Nodulate Parapiptadenia rigida (Benth.) Brenan
Authors: Taulé Cecilia , Zabaleta María , Mareque Cintia , Platero Raúl , Sanjurjo Lucía , Sicardi Margarita , Frioni Lillian , Battistoni Federico , Fabiano Elena
Published: January 2012

Among the leguminous trees native to Uruguay,
Parapiptadenia rigida
(Angico), a Mimosoideae legume, is one of the most promising species for agroforestry. Like many other legumes, it is able to establish symbiotic associations with rhizobia and belongs to the group known as nitrogen-fixing trees, which are major components of agroforestry systems. Information about rhizobial symbionts for this genus is scarce, and thus, the aim of this work was to identify and characterize rhizobia associated with
P. rigida
. A collection of Angico-nodulating isolates was obtained, and 47 isolates were selected for genetic studies. According to enterobacterial repetitive intergenic consensus PCR patterns and restriction fragment length polymorphism analysis of their
nifH
and 16S rRNA genes, the isolates could be grouped into seven genotypes, including the genera
Burkholderia
,
Cupriavidus
, and
Rhizobium
, among which the
Burkholderia
genotypes were the predominant group. Phylogenetic studies of
nifH
,
nodA
, and
nodC
sequences from the
Burkholderia
and the
Cupriavidus
isolates indicated a close relationship of these genes with those from betaproteobacterial rhizobia (beta-rhizobia) rather than from alphaproteobacterial rhizobia (alpha-rhizobia). In addition, nodulation assays with representative isolates showed that while the
Cupriavidus
isolates were able to effectively nodulate
Mimosa pudica
, the
Burkholderia
isolates produced white and ineffective nodules on this host.
High-Resolution Two-Locus Clonal Typing of Extraintestinal Pathogenic Escherichia coli
Authors: Weissman Scott J. , Johnson James R. , Tchesnokova Veronika , Billig Mariya , Dykhuizen Daniel , Riddell Kim , Rogers Peggy , Qin Xuan , Butler-Wu Susan , Cookson Brad T. , Fang Ferric C. , Scholes Delia , Chattopadhyay Sujay , Sokurenko Evgeni
Published: January 2012

Multilocus sequence typing (MLST) is usually based on the sequencing of 5 to 8 housekeeping loci in the bacterial chromosome and has provided detailed descriptions of the population structure of bacterial species important to human health. However, even strains with identical MLST profiles (known as sequence types or STs) may possess distinct genotypes, which enable different eco- or pathotypic lifestyles. Here we describe a two-locus, sequence-based typing scheme for
Escherichia coli
that utilizes a 489-nucleotide (nt) internal fragment of
fimH
(encoding the type 1 fimbrial adhesin) and the 469-nt internal
fumC
fragment used in standard MLST. Based on sequence typing of 191 model commensal and pathogenic isolates plus 853 freshly isolated clinical
E. coli
strains, this 2-locus approach—which we call CH (
fum

C

/
fim

H

) typing—consistently yielded more haplotypes than standard 7-locus MLST, splitting large STs into multiple clonal subgroups and often distinguishing different within-ST eco- and pathotypes. Furthermore, specific CH profiles corresponded to specific STs, or ST complexes, with 95% accuracy, allowing excellent prediction of MLST-based profiles. Thus, 2-locus CH typing provides a genotyping tool for molecular epidemiology analysis that is more economical than standard 7-locus MLST but has superior clonal discrimination power and, at the same time, corresponds closely to MLST-based clonal groupings.
Distribution of Intact and Core Membrane Lipids of Archaeal Glycerol Dialkyl Glycerol Tetraethers among Size-Fractionated Particulate Organic Matter in Hood Canal, Puget Sound
Authors: Ingalls Anitra E. , Huguet Carme , Truxal Laura T.
Published: January 2012

There is great interest in the membrane lipids of archaea (glycerol dialkyl glycerol tetraethers [GDGTs]) as tracers of archaeal biomass because of their utility as paleoproxies and because of the biogeochemical importance of archaea. While core GDGTs (formed by hydrolysis of polar head groups of intact GDGTs after cell death) are appropriate for paleostudies, they have also been used to trace archaeal populations. Also, despite the small size (0.2 by 0.7 μm) of cultivated marine archaea, 0.7-μm glass-fiber filters (GFFs) are typically used to collect GDGTs from natural waters. We quantified both core and intact GDGTs in free-living (0.2- to 0.7-μm), suspended (0.7- to 60-μm), and aggregate (>60-μm) particle size fractions in Puget Sound (Washington State). On average, the free-living fraction contained 36% of total GDGTs, 90% of which were intact. The intermediate-size fraction contained 62% of GDGTs, and 29% of these were intact. The aggregate fraction contained 2% of the total GDGT pool, and 29% of these were intact. Our results demonstrate that intact GDGTs are largely in the free-living fraction. Because only intact GDGTs are present in living cells, protocols that target this size fraction and analyze the intact GDGT pool are necessary to track living populations in marine waters. Core GDGT enrichment in larger-size fractions indicates that archaeal biomass may quickly become attached or entrained in particles once the archaea are dead or dying. While the concentrations of the two pools were generally not correlated, the similar sizes of the core and intact GDGT pools suggest that core GDGTs are removed from the water column on timescales similar to those of cell replication, on timescales of days to weeks.
Comparison of Gull Feces-Specific Assays Targeting the 16S rRNA Genes of Catellicoccus marimammalium and Streptococcus spp
Authors: Ryu Hodon , Griffith John F. , Khan Izhar U. H. , Hill Stephen , Edge Thomas A. , Toledo-Hernandez Carlos , Gonzalez-Nieves Joel , Santo Domingo Jorge
Published: January 2012

Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting
Streptococcus
spp. (gull3) and a hydrolysis TaqMan assay targeting
Catellicoccus marimammalium
(gull4). The objectives of this study were to compare the host specificity of a previous
C. marimammalium
qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (
n
= 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (
n
= 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (
n
= 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.
Counting Legionella Cells within Single Amoeba Host Cells
Authors: Buse Helen Y. , Ashbolt Nicholas J.
Published: January 2012

Here we present the first attempt to quantify
Legionella pneumophila
cell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturable
L. pneumophila
cells grown within
Acanthamoeba polyphaga
and
Naegleria fowleri
were 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite
−1
, respectively.
Regulation of Bacteriocin Production and Cell Death by the VicRK Signaling System in Streptococcus mutans
Authors: Senadheera D. B. , Cordova M. , Ayala E. A. , Chávez de Paz L. E. , Singh K. , Downey J. S. , Svensäter G. , Goodman S. D. , Cvitkovitch D. G.
Published: January 2012

The VicRK two-component signaling system modulates biofilm formation, genetic competence, and stress tolerance in
Streptococcus mutans
. We show here that the VicRK modulates bacteriocin production and cell viability, in part by direct modulation of competence-stimulating peptide (CSP) production in
S. mutans
. Global transcriptome and real-time transcriptional analysis of the VicK-deficient mutant (SmuvicK) revealed significant modulation of several bacteriocin-related loci, including
nlmAB
,
nlmC
, and
nlmD
(
P
< 0.001), suggesting a role for the VicRK in producing mutacins IV, V, and VI. Bacteriocin overlay assays revealed an altered ability of the
vic
mutants to kill related species. Since a well-conserved VicR binding site (TGTWAH-N
5
-TGTWAH) was identified within the
comC
coding region, we confirmed VicR binding to this sequence using DNA footprinting. Overexpression of the
vic
operon caused growth-phase-dependent repression of
comC
,
comDE
, and
comX.
In the
vic
mutants, transcription of
nlmC/cipB
encoding mutacin V, previously linked to CSP-dependent cell lysis, as well as expression of its putative immunity factor encoded by
immB
, were significantly affected relative to the wild type (
P
< 0.05). In contrast to previous reports that proposed a hyper-resistant phenotype for the VicK mutant in cell viability, the release of extracellular genomic DNA was significantly enhanced in SmuvicK (
P
< 0.05), likely as a result of increased autolysis compared with the parent. The drastic influence of VicRK on cell viability was also demonstrated using
vic
mutant biofilms. Taken together, we have identified a novel regulatory link between the VicRK and ComDE systems to modulate bacteriocin production and cell viability of
S. mutans
.
Characterization and Functional Analysis of
atl
, a Novel Gene Encoding Autolysin in Streptococcus suis
Authors: Ju Cun-Xiang , Gu Hong-Wei , Lu Cheng-Ping
Published: January 2012

Streptococcus suis
serotype 2 (
S. suis
2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated
atl
and encoding a major autolysin of
S. suis
2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved
N
-acetylmuramoyl-
l
-alanine amidase domain. Recombinant Atl was expressed in
Escherichia coli
, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the
atl
inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the
atl
mutant was reduced ∼30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the
atl
mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of
atl
inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.
Legionella pneumophila LidA Affects Nucleotide Binding and Activity of the Host GTPase Rab1
Authors: Neunuebel M. Ramona , Mohammadi Sina , Jarnik Michal , Machner Matthias P.
Published: January 2012

Legionella pneumophila
, the causative agent of a severe pneumonia known as Legionnaires' disease, intercepts material from host cell membrane transport pathways to create a specialized vacuolar compartment that supports bacterial replication. Delivery of bacterial effector proteins into the host cell requires the Dot/Icm type IV secretion system. Several effectors, including SidM, SidD, and LepB, were shown to target the early secretory pathway by manipulating the activity of the host GTPase Rab1. While the function of these effectors has been well characterized, the role of another Rab1-interacting protein from
L. pneumophila
, the effector protein LidA, is poorly understood. Here, we show that LidA binding to Rab1 stabilized the Rab1-guanosine nucleotide complex, protecting it from inactivation by GTPase-activating proteins (GAPs) and from nucleotide extraction. The protective effect of LidA on the Rab1-guanine nucleotide complex was concentration dependent, consistent with a 1:1 stoichiometry of the LidA-Rab1 complex. The central coiled-coil region of LidA was sufficient for Rab1 binding and to prevent GAP-mediated inactivation or nucleotide extraction from Rab1. In addition, the central region mediated binding to phosphatidylinositol 3-phosphate and other phosphoinositides. When bound to Rab1, LidA interfered with the covalent modification of Rab1 by phosphocholination or AMPylation, and it also blocked de-AMPylation of Rab1 by SidD and dephosphocholination by Lem3. Based on these findings, we propose a role for LidA in bridging the membrane of the
Legionella
-containing vacuole (LCV) with that of secretory transport vesicles surrounding the LCV.
Physiology of Resistant Deinococcus geothermalis Bacterium Aerobically Cultivated in Low-Manganese Medium
Authors: Liedert Christina , Peltola Minna , Bernhardt Jörg , Neubauer Peter , Salkinoja-Salonen Mirja
Published: January 2012

This dynamic proteome study describes the physiology of growth and survival of
Deinococcus geothermalis
, in conditions simulating paper machine waters being aerobic, warm, and low in carbon and manganese. The industrial environment of this species differs from its natural habitats, geothermal springs and deep ocean subsurfaces, by being highly exposed to oxygen. Quantitative proteome analysis using two-dimensional gel electrophoresis and bioinformatic tools showed expression change for 165 proteins, from which 47 were assigned to a function. We propose that
D. geothermalis
grew and survived in aerobic conditions by channeling central carbon metabolism to pathways where mainly NADPH rather than NADH was retrieved from the carbon source. A major part of the carbon substrate was converted into succinate, which was not a fermentation product but likely served combating reactive oxygen species (ROS). Transition from growth to nongrowth resulted in downregulation of the oxidative phosphorylation observed as reduced expression of V-type ATPase responsible for ATP synthesis in
D. geothermalis
. The battle against oxidative stress was seen as upregulation of superoxide dismutase (Mn dependent) and catalase, as well as several protein repair enzymes, including FeS cluster assembly proteins of the iron-sulfur cluster assembly protein system, peptidylprolyl isomerase, and chaperones. Addition of soluble Mn reinitiated respiration and proliferation with concomitant acidification, indicating that aerobic metabolism was restricted by access to manganese. We conclude that
D. geothermalis
prefers to combat ROS using manganese-dependent enzymes, but when manganese is not available central carbon metabolism is used to produce ROS neutralizing metabolites at the expense of high utilization of carbon substrate.
Wave intensity analysis of para-aortic counterpulsation
Authors: Lu Pong-Jeu , Yang Chi-Fu Jeffrey , Wu Meng-Yu , Hung Chun-Hao , Chan Ming-Yao , Hsu Tzu-Cheng
Published: January 2012

Wave intensity analysis (WIA) was used to delineate and maximize the efficacy of a newly developed para-aortic blood pump (PABP). The intra-aortic balloon pump (IABP) was employed as the comparison benchmark. Acute porcine experiments using eight pigs, randomly divided into IABP ( n = 4) and PABP ( n = 4) groups, were conducted to compare the characteristics of intra- and para-aortic counterpulsation. We measured pressure and velocity with probes installed in the left anterior descending coronary artery and aorta, during and without PABP assistance. Wave intensity for aortic and left coronary waves were derived from pressure and flow measurements with synchronization correction applied. To achieve maximized support efficacy, deflation timings ranging from 25 ms ahead of to 35 ms after the R-wave were tested. Similar to those associated with IABP counterpulsation, the PABP-generated backward-traveling waves predominantly drove aortic and coronary blood flows. However, in contrast with IABP counterpulsation, the nonocclusive nature of the PABP allowed systolic unloading to be delayed into early systole, which resulted in near elimination of coronary blood steal without diminution of systolic left ventricular ejection wave intensities. WIA can elucidate subtleties among different counterpulsatile support means with high sensitivity. Total accelerating wave intensity (TAWI), which was defined as the sum of the time integration of accelerated parts of the positive and negative wave intensities, was used to quantify counterpulsation efficacy. In general, the larger the TAWI gain, the better the counter-pulsatile support efficacy. However, when PABP deflation timings were delayed to after the R-wave, the TAWI was found to be inversely correlated with coronary perfusion. In this delayed deflation timing setting, greater wave cancellation occurred, which led to decreased TAWI but increased coronary perfusion attributed to blood regurgitation reduction.
The renin-angiotensin system: a target of and contributor to dyslipidemias, altered glucose homeostasis, and hypertension of the metabolic syndrome
Authors: Putnam Kelly , Shoemaker Robin , Yiannikouris Frederique , Cassis Lisa A.
Published: January 2012

The renin-angiotensin system (RAS) is an important therapeutic target in the treatment of hypertension. Obesity has emerged as a primary contributor to essential hypertension in the United States and clusters with other metabolic disorders (hyperglycemia, hypertension, high triglycerides, low HDL cholesterol) defined within the metabolic syndrome. In addition to hypertension, RAS blockade may also serve as an effective treatment strategy to control impaired glucose and insulin tolerance and dyslipidemias in patients with the metabolic syndrome. Hyperglycemia, insulin resistance, and/or specific cholesterol metabolites have been demonstrated to activate components required for the synthesis [angiotensinogen, renin, angiotensin-converting enzyme (ACE)], degradation (ACE2), or responsiveness (angiotensin II type 1 receptors, Mas receptors) to angiotensin peptides in cell types (e.g., pancreatic islet cells, adipocytes, macrophages) that mediate specific disorders of the metabolic syndrome. An activated local RAS in these cell types may contribute to dysregulated function by promoting oxidative stress, apoptosis, and inflammation. This review will discuss data demonstrating the regulation of components of the RAS by cholesterol and its metabolites, glucose, and/or insulin in cell types implicated in disorders of the metabolic syndrome. In addition, we discuss data supporting a role for an activated local RAS in dyslipidemias and glucose intolerance/insulin resistance and the development of hypertension in the metabolic syndrome. Identification of an activated RAS as a common thread contributing to several disorders of the metabolic syndrome makes the use of angiotensin receptor blockers and ACE inhibitors an intriguing and novel option for multisymptom treatment.
Intermedin elicits a negative inotropic effect in rat papillary muscles mediated by endothelial-derived nitric oxide
Authors: Pires Ana Luísa , Pinho Marta , Sena Cristina Maria , Seica Raquel , Leite-Moreira Adelino F.
Published: January 2012

Intermedin (IMD) is a novel vasoactive peptide from the calcitonin gene-related peptide (CGRP) implicated in cardiac regulation, yet the contractile effects of IMD remain controversial, since previous studies in vivo and isolated cardiomyocytes documented contradictory results. We hypothesized cardiac endothelial cells involvement in IMD modulation of cardiac function as an explanation for these opposing observations. With this in mind, we investigated the direct action of increasing concentrations of IMD (10−8 to 10−6M) on myocardial performance parameters in rat left ventricular (LV) papillary muscles with and without endocardial endothelium (EE) and in presence of receptor antagonists and intracellular pathways inhibitors. In LV papillary muscles with intact EE, IMD induced a concentration-dependent negative inotropic action (%decrease relative to baseline, at IMD concentration of 10−6M, active tension of 14 ± 4%, and maximum velocity of tension rise of 10 ± 4%). These effects were blunted by EE removal, AM receptor antagonist (AM22–52), and CGRP receptor antagonist (CGRP8–37). Additionally, nitric oxide (NO) synthase inhibition with NG-nitro-l-arginine (l-NAME) in muscles with and without EE and guanylyl cyclase inhibition with {1 H-[1,2,4]oxadiazole-[4,4-a]-quinoxalin-1-one} not only blunted the negative inotropic action of IMD but also unmasked IMD-positive inotropic effect dependent on CGRP receptor PKA activation. Western blot quantification of phosphorylated cardiac troponin I (P-cTnI) in IMD-treated papillary muscles revealed a significant increase in P-cTnI when compared with untreated muscles, while in l-NAME-pretreated papillary muscles IMD failed to increase P-cTnI. Finally, we found that stimulation of both EE and microvascular endothelial cells with IMD significantly increased NO production by 40 ± 3 and 38 ± 3%, respectively, suggesting the role of cardiac endothelial cells in NO production upon IMD stimulation. Our findings establish IMD negative inotropic effect in isolated myocardium due to NO/cGMP pathway activation with concomitant thin myofilament desensitization by increase in cTnI phosphorylation and provide a coherent explanation for the previously reported contradictory results.
Effect of alveolar epithelial cell plasticity on the regulation of GM-CSF expression
Authors: Mir-Kasimov Mustafa , Sturrock Anne , McManus Michael , Paine Robert
Published: January 2012

Local pulmonary expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically important for defense of the pulmonary alveolar space. It is required for surfactant homeostasis and pulmonary innate immune responses and is protective against lung injury and aberrant repair. Alveolar epithelial cells (AEC) are a major source of GM-CSF; however, the control of homeostatic expression of GM-CSF is incompletely characterized. Increasing evidence suggests considerable plasticity of expression of AEC phenotypic characteristics. We tested the hypothesis that this plasticity extends to regulation of expression of GM-CSF using 1) MLE-12 cells (a commonly used murine cell line expressing some features of normal type II AEC, 2) primary murine AEC incubated under standard conditions [resulting in rapid spreading and loss of surfactant protein C (SP-C) expression with induction of the putative type I cell marker (T1α)], or 3) primary murine AEC on a hyaluronic acid/collagen matrix in defined medium, resulting in preservation of SP-C expression. AEC in standard cultures constitutively express abundant GM-CSF, with further induction in response to IL-1β but little response to TNF-α. In contrast, primary cells cultured to preserve SP-C expression and MLE-12 cells both express little GM-CSF constitutively, with significant induction in response to TNF-α and limited response to IL-1β. We conclude that constitutive and cytokine-induced expression of GM-CSF by AEC varies in concert with other cellular phenotypic characteristics. These changes may have important implications both for the maintenance of normal pulmonary homeostasis and for the process of repair following lung injury.
BMP type II receptor deficiency confers resistance to growth inhibition by TGF-β in pulmonary artery smooth muscle cells: role of proinflammatory cytokines
Authors: Davies Rachel J. , Holmes Alan M. , Deighton John , Long Lu , Yang Xudong , Barker Lucy , Walker Christoph , Budd David C. , Upton Paul D. , Morrell Nicholas W.
Published: January 2012

Mutations in the bone morphogenetic protein (BMP) type II receptor (BMPR-II) underlie most cases of heritable pulmonary arterial hypertension (HPAH) and a significant proportion of sporadic cases. Pulmonary artery smooth muscle cells (PASMCs) from patients with pulmonary arterial hypertension (PAH) not only exhibit attenuated growth suppression by BMPs, but an abnormal mitogenic response to transforming growth factor (TGF)-β1. We sought to define the mechanism underlying this loss of the antiproliferative effects of TGF-β1 in BMPR-II-deficient PASMCs. The effect of TGF-β1 on PASMC proliferation was characterized in three different models of BMPR-II dysfunction: 1) HPAH PASMCs, 2) Bmpr2+/− mouse PASMCs, and 3) control human PASMCs transfected with BMPR-II small interfering RNA. BMPR-II reduction consistently conferred insensitivity to growth inhibition by TGF-β1. This was not associated with altered canonical TGF-β1/Smad signaling but was associated with a secreted factor. Microarray analysis revealed that the transcriptional responses to TGF-β1 differed between control and HPAH PASMCs, particularly regarding genes associated with interleukins and inflammation. HPAH PASMCs exhibited enhanced IL-6 and IL-8 induction by TGF-β1, an effect reversed by NF-κB inhibition. Moreover, neutralizing antibodies to IL-6 or IL-8 restored the antiproliferative effect of TGF-β1 in HPAH PASMCs. This study establishes that BMPR-II deficiency leads to failed growth suppression by TGF-β1 in PASMCs. This effect is Smad-independent but is associated with inappropriately altered NF-κB signaling and enhanced induction of IL-6 and IL-8 expression. Our study provides a rationale to test anti-interleukin therapies as an intervention to neutralize this inappropriate response and restore the antiproliferative response to TGF-β1.
Immunochemotherapy with Intensive Consolidation for Primary CNS Lymphoma: A Pilot Study and Prognostic Assessment by Diffusion-Weighted MRI
Authors: Wieduwilt Matthew J. , Valles Francisco , Issa Samar , Behler Caroline M. , Hwang James , McDermott Michael , Treseler Patrick , O'Brien Joan , Shuman Marc A. , Cha Soonmee , Damon Lloyd E. , Rubenstein James L.
Published: January 2012

Purpose: We evaluated a novel therapy for primary central nervous system lymphoma (PCNSL) with induction immunochemotherapy with high-dose methotrexate, temozolomide, and rituximab (MT-R) followed by intensive consolidation with infusional etoposide and high-dose cytarabine (EA). In addition, we evaluated the prognostic value of the minimum apparent diffusion coefficient (ADCmin) derived from diffusion-weighted MRI (DW-MRI) in patients treated with this regimen.

Experimental Design: Thirty-one patients (median age, 61 years; median Karnofsky performance score, 60) received induction with methotrexate every 14 days for 8 planned cycles. Rituximab was administered the first 6 cycles and temozolomide administered on odd-numbered cycles. Patients with responsive or stable central nervous system (CNS) disease received EA consolidation. Pretreatment DW-MRI was used to calculate the ADCmin of contrast-enhancing lesions.

Results: The complete response rate for MT-R induction was 52%. At a median follow-up of 79 months, the 2-year progression-free and overall survival were 45% and 58%, respectively. For patients receiving EA consolidation, the 2-year progression-free and overall survival were 78% and 93%, respectively. EA consolidation was also effective in an additional 3 patients who presented with synchronous CNS and systemic lymphoma. Tumor ADCmin less than 384 × 10–6 mm2/s was significantly associated with shorter progression-free and overall survival.

Conclusions: MT-R induction was effective and well tolerated. MT-R followed by EA consolidation yielded progression-free and overall survival outcomes comparable to regimens with chemotherapy followed by whole-brain radiotherapy consolidation but without evidence of neurotoxicity. Tumor ADCmin derived from DW-MRI provided better prognostic information for PCNSL patients treated with the MTR-EA regimen than established clinical risk scores. Clin Cancer Res; 18(4); 1146–55. ©2012 AACR.
Notch Signaling Promotes Growth and Invasion in Uveal Melanoma
Authors: Asnaghi Laura , Ebrahimi Katayoon B. , Schreck Karisa C. , Bar Eli E. , Coonfield Michael L. , Bell W. Robert , Handa James , Merbs Shannath L. , Harbour J. William , Eberhart Charles G.
Published: January 2012

Purpose: To determine whether uveal melanoma, the most common primary intraocular malignancy in adults, requires Notch activity for growth and metastasis.

Experimental Design: Expression of Notch pathway members was characterized in primary tumor samples and in cell lines, along with the effects of Notch inhibition or activation on tumor growth and invasion.

Results: Notch receptors, ligands, and targets were expressed in all five cell lines examined and in 30 primary uveal melanoma samples. Interestingly, the three lines with high levels of baseline pathway activity (OCM1, OCM3, and OCM8) had their growth reduced by pharmacologic Notch blockade using the γ-secretase inhibitor (GSI) MRK003. In contrast, two uveal melanoma lines (Mel285 and Mel290) with very low expression of Notch targets were insensitive to the GSI. Constitutively active forms of Notch1 and Notch2 promoted growth of uveal melanoma cultures and were able to rescue the inhibitory effects of GSI. MRK003 treatment also inhibited anchorage-independent clonogenic growth and cell invasion and reduced phosphorylation levels of STAT3 and extracellular signal-regulated kinase (Erk)1/2. Suppression of canonical Notch activity using short hairpin RNA targeting Notch2 or CBF1 was also able to reduce tumor growth and invasion. Finally, intraocular xenograft growth was significantly decreased by GSI treatment.

Conclusion: Our findings suggest that Notch plays an important role in inducing proliferation and invasion in uveal melanoma and that inhibiting this pathway may be effective in preventing tumor growth and metastasis. Clin Cancer Res; 18(3); 654–65. ©2012 AACR.
Regulation of CD20 in Rituximab-Resistant Cell Lines and B-cell Non-Hodgkin Lymphoma
Authors: Tsai Ping-Chiao , Hernandez-Ilizaliturri Francisco J. , Bangia Naveen , Olejniczak Scott H. , Czuczman Myron S.
Published: January 2012

Purpose: The aim of this research was to further investigate the contribution of CD20 antigen expression to rituximab activity and define the mechanisms responsible for CD20 downregulation in rituximab-resistant cell lines (RRCL).

Experimental Design: Rituximab-sensitive cell lines, RRCL, and primary neoplastic B cells were evaluated by chromium-51 release assays, ImageStream image analysis, immunohistochemical staining, flow cytometric analysis, CD20 knockdown, promoter activity, chromatin immunoprecipitation (ChIP) analysis of CD20 promoter, and CD20 plasmid transfection experiments to identify mechanisms associated with CD20 regulation in RRCL.

Results: RRCL exhibited a gradual loss of CD20 surface expression with repeated exposure to rituximab. We identified a CD20 antigen surface threshold level required for effective rituximab-associated complement-mediated cytotoxicity (CMC). However, a direct correlation between CD20 surface expression and rituximab-CMC was observed only in rituximab-sensitive cell lines. CD20 promoter activity was decreased in RRCL. Detailed analysis of various CD20 promoter fragments suggested a lack of positive regulatory factors in RRCL. ChIP analysis showed reduced binding of several key positive regulatory proteins on CD20 promoter in RRCL. Interleukin-4 (IL-4) induced higher CD20 promoter activity and CD20 expression but modestly improved rituximab activity in RRCL and in primary B-cell lymphoma cells. Forced CD20 expression restored cytoplasmic but not surface CD20, suggesting the existence of a defect in CD20 protein transport in RRCL.

Conclusions: We identified several mechanisms that alter CD20 expression in RRCL and showed that, whereas CD20 expression is important for rituximab activity, additional factors likely contribute to rituximab sensitivity in B-cell lymphoma. Clin Cancer Res; 18(4); 1039–50. ©2012 AACR.
Phase II Trial of Temozolomide in Patients with Relapsed Sensitive or Refractory Small Cell Lung Cancer, with Assessment of Methylguanine-DNA Methyltransferase as a Potential Biomarker
Authors: Pietanza M. Catherine , Kadota Kyuichi , Huberman Kety , Sima Camelia S. , Fiore John J. , Sumner Dyana K. , Travis William D. , Heguy Adriana , Ginsberg Michelle S. , Holodny Andrei I. , Chan Timothy A. , Rizvi Naiyer A. , Azzoli Christopher G. , Riely Gregory J. , Kris Mark G. , Krug Lee M.
Published: January 2012

Purpose: This phase II study was conducted to assess the efficacy of temozolomide in patients with relapsed small cell lung cancer (SCLC).

Experimental Design: Patients with disease progression after one or two prior chemotherapy regimens received temozolomide at 75 mg/m2/d for 21 days of a 28-day cycle. The primary endpoint was the overall response rate [ORR; complete response (CR) plus partial response (PR)], which was evaluated separately in sensitive and refractory cohorts. In the available tissue, we assessed O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status by PCR and MGMT expression by immunohistochemistry.

Results: Sixty-four patients were accrued: 48 patients in the sensitive cohort and 16 in the refractory group. One CR and 10 PRs were noted in sensitive patients [ORR, 23%; 95% confidence interval (CI), 12%–37%]. Two PRs were seen in the refractory cohort (ORR, 13%; 95% CI, 2%–38%). As second- and third-line treatment, the ORR was 22% (95% CI, 9%–40%) and 19% (95% CI, 7%–36%), respectively. Among patients with target brain lesions, 38% had a CR or PR (95% CI, 14%–68%). Grade ≥3 thrombocytopenia and neutropenia were observed in nine patients (14%). A greater number of cases with methylated MGMT had a response compared to those with unmethylated MGMT (38% vs. 7%; P = 0.08).

Conclusion: Temozolomide has activity in relapsed SCLC, particularly for brain metastases. Response to temozolomide may correlate with MGMT methylation in SCLC. Clin Cancer Res; 18(4); 1138–45. ©2012 AACR.
Changes in Keratin Expression during Metastatic Progression of Breast Cancer: Impact on the Detection of Circulating Tumor Cells
Authors: Joosse Simon A. , Hannemann Juliane , Spötter Julia , Bauche Andreas , Andreas Antje , Müller Volkmar , Pantel Klaus
Published: January 2012

Purpose: Circulating tumor cells (CTC) might function as early markers for breast cancer metastasis or monitoring therapy efficacy. Enrichment and identification of CTCs are based on epithelial markers that might be modulated during epithelial–mesenchymal transition. Little is known about the expression of keratins in CTCs and whether all CTCs can be detected with antibodies directed against a limited panel of keratins.

Experimental Design: Protein expression of keratin 2, 4–10, 13–16, 18, and 19 were assessed by a cocktail of antibodies (C11, AE1, AE3, and K7) and keratin antibodies C11 and A45-B/B3 alone in 11 breast cancer cell lines and 50 primary breast carcinomas and their lymph node metastases. Furthermore, CTCs were assessed in blood of 70 metastatic breast cancer patients.

Results: Claudin-low cell lines did not show expression of normal breast epithelial keratins but were positive for K14 and K16, detected by the cocktail only. Primary breast carcinomas showed changes in keratin expression during metastatic progression to the lymph nodes. In 35 of 70 patients CTCs were identified, of which 83%, 40%, and 57% were identified by the cocktail, C11 and A45-B/B3, respectively. Identification of CTCs by the cocktail was associated with shorter survival (P < 0.01). In silico analyses revealed association between KRT16 expression and shorter relapse-free survival in metastatic breast cancer.

Conclusion: Breast cancer cells show a complex pattern of keratin expression with potential biologic relevance. Individual keratin antibodies recognizing only a limited set of keratins inherit the risk to miss biologically relevant CTCs in cancer patients, and antibody cocktails including these keratins are therefore recommended. Clin Cancer Res; 18(4); 993–1003. ©2012 AACR.
Combination of a Novel Gene Expression Signature with a Clinical Nomogram Improves the Prediction of Survival in High-Risk Bladder Cancer
Authors: Riester Markus , Taylor Jennifer M. , Feifer Andrew , Koppie Theresa , Rosenberg Jonathan E. , Downey Robert J. , Bochner Bernard H. , Michor Franziska
Published: January 2012

Purpose: We aimed to validate and improve prognostic signatures for high-risk urothelial carcinoma of the bladder.

Experimental Design: We evaluated microarray data from 93 patients with bladder cancer managed by radical cystectomy to determine gene expression patterns associated with clinical and prognostic variables. We compared our results with published bladder cancer microarray data sets comprising 578 additional patients and with 49 published gene signatures from multiple cancer types. Hierarchical clustering was utilized to identify subtypes associated with differences in survival. We then investigated whether the addition of survival-associated gene expression information to a validated postcystectomy nomogram utilizing clinical and pathologic variables improves prediction of recurrence.

Results: Multiple markers for muscle invasive disease with highly significant expression differences in multiple data sets were identified, such as fibronectin 1 (FN1), NNMT, POSTN, and SMAD6. We identified signatures associated with pathologic stage and the likelihood of developing metastasis and death from bladder cancer, as well as with two distinct clustering subtypes of bladder cancer. Our novel signature correlated with overall survival in multiple independent data sets, significantly improving the prediction concordance of standard staging in all data sets [mean ΔC-statistic: 0.14; 95% confidence interval (CI), 0.01–0.27; P < 0.001]. Tested in our patient cohort, it significantly enhanced the performance of a postoperative survival nomogram (ΔC-statistic: 0.08, 95% CI, −0.04–0.20; P < 0.005).

Conclusions: Prognostic information obtained from gene expression data can aid in posttreatment prediction of bladder cancer recurrence. Our findings require further validation in external cohorts and prospectively in a clinical trial setting. Clin Cancer Res; 18(5); 1323–33. ©2012 AACR.
Detection of TIMP3 Promoter Hypermethylation in Salivary Rinse as an Independent Predictor of Local Recurrence-Free Survival in Head and Neck Cancer
Authors: Sun Wenyue , Zaboli David , Wang Hao , Liu Yan , Arnaoutakis Demetri , Khan Tanbir , Khan Zubair , Koch Wayne M. , Califano Joseph A.
Published: January 2012

Purpose: To validate a panel of methylation-based salivary rinse biomarkers (P16, CCNA1, DCC, TIMP3, MGMT, DAPK, and MINT31) previously shown to be independently associated with poor overall survival and local recurrence in a larger, separate cohort of patients with head and neck squamous cell carcinoma (HNSCC).

Experimental Design: One hundred ninety-seven patients were included. All pretreatment saliva DNA samples were evaluated for the methylation status of the gene promoters by quantitative methylation-specific PCR. The main outcome measures were overall survival, local recurrence-free survival, and disease-free survival.

Results: In univariate analyses, the detection of hypermethylation of CCNA1, MGMT, and MINT31 was significantly associated with poor overall survival; the detection of hypermethylation of TIMP3 was significantly associated with local recurrence-free survival; and the detection of hypermethylation of MINT31 was significantly associated with poor disease-free survival. In multivariate analyses, detection of hypermethylation at any single marker was not predictive of overall survival in patients with HNSCC; detection of hypermethylation of TIMP3 in salivary rinse had an independent, significant association with local recurrence-free survival (HR = 2.51; 95% CI: 1.10–5.68); and none of the studied markers was significantly associated with disease-free survival.

Conclusion: The detection of promoter hypermethylation of the seven genes in salivary rinse as an independent prognostic indicator of overall survival in patients with HNSCC was not validated. Detection of promoter hypermethylation of TIMP3 in pretreatment salivary rinse is independently associated with local recurrence-free survival in patients with HNSCC and may be a valuable salivary rinse biomarker for HNSCC recurrence. Clin Cancer Res; 18(4); 1082–91. ©2012 AACR.