bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of Highly Heat-Resistant Listeria monocytogenes Variants by Use of a Kinetic Modeling-Based Sampling Scheme
Authors: Van Boeijen Ineke K. H. , Francke Christof , Moezelaar Roy , Abee Tjakko , Zwietering Marcel H.
Published: February 2011

Stable high-hydrostatic-pressure (HHP)-resistant
Listeria monocytogenes
LO28 variants were previously isolated and characterized. These HHP variants were also more resistant to heat. In addition, nonlinear heat inactivation kinetics pointed toward the existence of heat-resistant variants, although these could not be isolated so far. In this study, we used kinetic modeling of inactivation curves of two isolated HHP variants and their wild type, and this revealed that the probability of finding resistant variants should depend on the nature of the inactivation treatment and the time of exposure. At specific heat and HHP conditions, resistant LO28 and EGDe variants were indeed isolated. Resistant LO28 variants were even isolated after a heat inactivation at 72°C in milk, and these variants showed high resistance to standard pasteurization conditions. The increased resistance of part of the isolated LO28 and EGDe variants was due to mutations in their
ctsR
genes. For the variants whose
ctsR
genes and upstream regions were not altered, the mechanisms leading to increased resistance remain to be elucidated. This research showed the strength of kinetic modeling in unraveling the causes of nonlinear inactivation and facilitating the isolation of heat-resistant
L. monocytogenes
variants.
Moonlighting Role of a Poly-γ-Glutamate Synthetase Component from Bacillus subtilis: Insight into Novel Extrachromosomal DNA Maintenance
Authors: Yamashiro Daisuke , Minouchi Yutaka , Ashiuchi Makoto
Published: February 2011

The
Bacillus subtilis
structural gene
pgsE
was investigated as a tool for extrachromosomal DNA maintenance (EDM). It ameliorated the stability of high-copy-number vectors, regardless of whether they were derived from rolling-cycle or theta-mode replicons, without any selective pressure. This unique EDM phenomenon may occur via a
trans
-acting mechanism.
Comparative Analysis of Antimicrobial Activities of Valinomycin and Cereulide, the Bacillus cereus Emetic Toxin
Authors: Tempelaars Marcel H. , Rodrigues Susana , Abee Tjakko
Published: February 2011

Cereulide and valinomycin are highly similar cyclic dodecadepsipeptides with potassium ionophoric properties. Cereulide, produced by members of the
Bacillus cereus
group, is known mostly as emetic toxin, and no ecological function has been assigned. A comparative analysis of the antimicrobial activity of valinomycin produced by
Streptomyces
spp. and cereulide was performed at a pH range of pH 5.5 to pH 9.5, under anaerobic and aerobic conditions. Both compounds display pH-dependent activity against selected Gram-positive bacteria, including
Staphylococcus aureus
,
Listeria innocua
,
Listeria monocytogenes
,
Bacillus subtilis
, and
Bacillus cereus
ATCC 10987. Notably,
B. cereus
strain ATCC 14579 and the emetic
B. cereus
strains F4810/72 and A529 showed reduced sensitivity to both compounds, with the latter two strains displaying full resistance to cereulide. Both compounds showed no activity against the selected Gram-negative bacteria. Antimicrobial activity against Gram-positive bacteria was highest at alkaline pH values, where the membrane potential (ΔΨ) is the main component of the proton motive force (PMF). Furthermore, inhibition of growth was observed in both aerobic and anaerobic conditions. Determination of the ΔΨ, using the membrane potential probe DiOC
2
(3) (in the presence of 50 mM KCl) in combination with flow cytometry, demonstrated for the first time the ability of cereulide to dissipate the ΔΨ in sensitive Gram-positive bacteria. The putative role of cereulide production in the ecology of emetic
B. cereus
is discussed.
Detecting Potentially Virulent Vibrio vulnificus Strains in Raw Oysters by Quantitative Loop-Mediated Isothermal Amplification
Authors: Han Feifei , Wang Fei , Ge Beilei
Published: February 2011

Vibrio vulnificus
is a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (
vcg
) have been used to distinguish between clinical and environmental
V. vulnificus
strains, with a strong association between clinical ones and the C sequence variant (
vcgC
). In this study,
vcgC
was selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulent
V. vulnificus
strains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulent
V. vulnificus
strain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 × 10
3
CFU/g of
V. vulnificus
ATCC 33815, while showing negative results for a nonvirulent
V. vulnificus
strain (515-4c2) spiked at 10
7
CFU/g. After 6 h of enrichment, the LAMP assay could detect 1 CFU/g of the virulent
V. vulnificus
strain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulent
V. vulnificus
strain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulent
V. vulnificus
in raw oysters with high speed, specificity, and sensitivity, which may facilitate better control of
V. vulnificus
risks associated with raw oyster consumption.
Mid1, a Mechanosensitive Calcium Ion Channel, Affects Growth, Development, and Ascospore Discharge in the Filamentous Fungus Gibberella zeae
Authors: Cavinder Brad , Hamam Ahmed , Lew Roger R. , Trail Frances
Published: February 2011

The role of Mid1, a stretch-activated ion channel capable of being permeated by calcium, in ascospore development and forcible discharge from asci was examined in the pathogenic fungusGibberella zeae(anamorphFusarium graminearum). The Δmid1mutants exhibited a >12-fold reduction in ascospore discharge activity and produced predominately abnormal two-celled ascospores with constricted and fragile septae. The vegetative growth rate of the mutants was ∼50% of the wild-type rate, and production of macroconidia was >10-fold lower than in the wild type. To better understand the role of calcium flux, Δmid1Δcch1double mutants were also examined, as Cch1, an L-type calcium ion channel, is associated with Mid1 inSaccharomyces cerevisiae. The phenotype of the Δmid1Δcch1double mutants was similar to but more severe than the phenotype of the Δmid1mutants for all categories. Potential and current-voltage measurements were taken in the vegetative hyphae of the Δmid1and Δcch1mutants and the wild type, and the measurements for all three strains were remarkably similar, indicating that neither protein contributes significantly to the overall electrical properties of the plasma membrane. Pathogenicity of the Δmid1and Δmid1Δcch1mutants on the host (wheat) was not affected by the mutations. Exogenous calcium supplementation partially restored the ascospore discharge and vegetative growth defects for all mutants, but abnormal ascospores were still produced. These results extend the known roles of Mid1 to ascospore development and forcible discharge. However,Neurospora crassaΔmid1mutants were also examined and did not exhibit defects in ascospore development or in ascospore discharge. In comparison to ion channels in other ascomycetes, Mid1 shows remarkable adaptability of roles, particularly with regard to niche-specific adaptation.
Role of B Regulatory Subunits of Protein Phosphatase Type 2A in Myosin II Assembly Control in Dictyostelium discoideum
Authors: Rai Vandana , Egelhoff Thomas T.
Published: February 2011

In
Dictyostelium discoideum
, myosin II resides predominantly in a soluble pool as the result of phosphorylation of the myosin heavy chain (MHC), and dephosphorylation of the MHC is required for myosin II filament assembly, recruitment to the cytoskeleton, and force production. Protein phosphatase type 2A (PP2A) was identified in earlier studies in
Dictyostelium
as a key biochemical activity that can drive MHC dephosphorylation. We report here gene targeting and cell biological studies addressing the roles of candidate PP2A B regulatory subunits (phr2aBα and phr2aBβ) in myosin II assembly control
in vivo
.
Dictyostelium
phr2aBα- and phr2aBβ-null cells show delayed development, reduction in the assembly of myosin II in cytoskeletal ghost assays, and defects in cytokinesis when grown in suspension compared to parental cell lines. These results demonstrate that the PP2A B subunits phr2aBα and phr2aBβ contribute to myosin II assembly control
in vivo
, with phr2aBα having the predominant role facilitating MHC dephosphorylation to facilitate filament assembly.
Conjugated Linoleic Acid Inhibits Hyphal Growth in Candida albicans by Modulating Ras1p Cellular Levels and Downregulating TEC1 Expression
Authors: Shareck Julie , Nantel André , Belhumeur Pierre
Published: February 2011

The polymorphic yeast
Candida albicans
exists in yeast and filamentous forms. Given that the morphogenetic switch coincides with the expression of many virulence factors, the yeast-to-hypha transition constitutes an attractive target for the development of new antifungal agents. Since an untapped therapeutic potential resides in small molecules that hinder
C. albicans
filamentation, we characterized the inhibitory effect of conjugated linoleic acid (CLA) on hyphal growth and addressed its mechanism of action. CLA inhibited hyphal growth in a dose-dependent fashion in both liquid and solid hypha-inducing media. The fatty acid blocked germ tube formation without affecting cellular growth rates. Global transcriptional profiling revealed that CLA downregulated the expression of hypha-specific genes and abrogated the induction of several regulators of hyphal growth, including
TEC1
,
UME6
,
RFG1
, and
RAS1
. However, neither
UME6
nor
RFG1
was necessary for CLA-mediated hyphal growth inhibition. Expression analysis showed that the downregulation of
TEC1
expression levels by CLA depended on
RAS1
. In addition, while
RAS1
transcript levels remained constant in CLA-treated cells, its protein levels declined with time. With the use of a strain expressing GFP-Ras1p, CLA treatment was also shown to affect Ras1p localization to the plasma membrane. These findings suggest that CLA inhibits hyphal growth by affecting the cellular localization of Ras1p and blocking the increase in
RAS1
mRNA and protein levels. Combined, these effects should prevent the induction of the Ras1p signaling pathway. This study provides the biological and molecular explanations that underlie CLA's ability to inhibit hyphal growth in
C. albicans
.
Molecular Cloning and Characterization of the
srdBCA
Operon, Encoding the Respiratory Selenate Reductase Complex, from the Selenate-Reducing Bacterium Bacillus selenatarsenatis SF-1
Authors: Kuroda Masashi , Yamashita Mitsuo , Miwa Emiko , Imao Kanako , Fujimoto Noriyuki , Ono Hisayo , Nagano Kouta , Sei Kazunari , Ike Michihiko
Published: February 2011

Previously, we isolated a selenate- and arsenate-reducing bacterium, designated strain SF-1, from selenium-contaminated sediment and identified it as a novel species,
Bacillus selenatarsenatis. B. selenatarsenatis
strain SF-1 independently reduces selenate to selenite, arsenate to arsenite, and nitrate to nitrite by anaerobic respiration. To identify the genes involved in selenate reduction, 17 selenate reduction-defective mutant strains were isolated from a mutant library generated by random insertion of transposon Tn
916
. Tn
916
was inserted into the same genome position in eight mutants, and the representative strain SF-1AM4 did not reduce selenate but did reduce nitrate and arsenate to the same extent as the wild-type strain. The disrupted gene was located in an operon composed of three genes designated
srdBCA
, which were predicted to encode a putative oxidoreductase complex by the BLASTX program. The plasmid vector pGEMsrdBCA, containing the
srdBCA
operon with its own promoter, conferred the phenotype of selenate reduction in
Escherichia coli
DH5α, although
E. coli
strains containing plasmids lacking any one or two of the open reading frames from
srdBCA
did not exhibit the selenate-reducing phenotype. Domain structure analysis of the deduced amino acid sequence revealed that SrdBCA had typical features of membrane-bound and molybdopterin-containing oxidoreductases. It was therefore proposed that the
srdBCA
operon encoded a respiratory selenate reductase complex. This is the first report of genes encoding selenate reductase in Gram-positive bacteria.
Mutation of a Broadly Conserved Operon (RL3499-RL3502) from Rhizobium leguminosarum Biovar
viciae
Causes Defects in Cell Morphology and Envelope Integrity
Authors: Vanderlinde Elizabeth M. , Magnus Samantha A. , Tambalo Dinah D. , Koval Susan F. , Yost Christopher K.
Published: February 2011

The bacterial cell envelope is of critical importance to the function and survival of the cell; it acts as a barrier against harmful toxins while allowing the flow of nutrients into the cell. It also serves as a point of physical contact between a bacterial cell and its host. Hence, the cell envelope of
Rhizobium leguminosarum
is critical to cell survival under both free-living and symbiotic conditions. Transposon mutagenesis of
R. leguminosarum
strain 3841 followed by a screen to isolate mutants with defective cell envelopes led to the identification of a novel conserved operon (RL3499-RL3502) consisting of a putative
moxR
-like AAA
+
ATPase, a hypothetical protein with a domain of unknown function (designated domain of unknown function 58), and two hypothetical transmembrane proteins. Mutation of genes within this operon resulted in increased sensitivity to membrane-disruptive agents such as detergents, hydrophobic antibiotics, and alkaline pH. On minimal media, the mutants retain their rod shape but are roughly 3 times larger than the wild type. On media containing glycine or peptides such as yeast extract, the mutants form large, distorted spheres and are incapable of sustained growth under these culture conditions. Expression of the operon is maximal during the stationary phase of growth and is reduced in a
chvG
mutant, indicating a role for this sensor kinase in regulation of the operon. Our findings provide the first functional insight into these genes of unknown function, suggesting a possible role in cell envelope development in
Rhizobium leguminosarum
. Given the broad conservation of these genes among the
Alphaproteobacteria
, the results of this study may also provide insight into the physiological role of these genes in other
Alphaproteobacteria
, including the animal pathogen
Brucella
.
Single-Gene Deletion Mutants of Escherichia coli with Altered Sensitivity to Bicyclomycin, an Inhibitor of Transcription Termination Factor Rho
Authors: Tran Lillian , van Baarsel Joshua A. , Washburn Robert S. , Gottesman Max E. , Miller Jeffrey H.
Published: February 2011

We have screened the entire KEIO collection of 3,985 single-gene knockouts in
Escherichia coli
for increased susceptibility or resistance to the antibiotic bicyclomycin (BCM), a potent inhibitor of the transcription termination factor Rho. We also compared the results to those of a recent study we conducted with a large set of antibiotics (A. Liu et al., Antimicrob. Agents Chemother.
54:
1393-1403, 2010). We find that deletions of many different types of genes increase sensitivity to BCM. Some of these are involved in multidrug sensitivity/resistance, whereas others are specific for BCM. Mutations in a number of DNA recombination and repair genes increase BCM sensitivity, indicating that DNA damage leading to single- and double-strand breaks is a downstream effect of Rho inhibition. MDS42, which is deleted for all cryptic prophages and insertion elements (G. Posfai et al., Science
312:
1044-1046, 2006), or W3102 deleted for the
rac
prophage-encoded
kil
gene, are partially resistant to BCM (C. J. Cardinale et al., Science
230:
935-938, 2008). Deletion of cryptic prophages also overcomes the increased BCM sensitivity in some but not all mutants examined here. Deletion of the
hns
gene renders the cell more sensitive to BCM even in the Δ
kil
or MDS42 background. This suggests that BCM activates additional modes of cell death independent of Kil and that these could provide a target to potentiate BCM killing.
An approach to recurrent aneurysms following endovascular coiling
Authors: Raymond Jean , Darsaut Tim E
Published: February 2011

Background

Evidence regarding the most appropriate management of aneurysm recurrences following coiling consists only of uncontrolled case series and the resulting expert opinions, which range from not following coiled patients at all to performing invasive angiography at regular intervals.

Methods

We discuss several current attitudes toward how to manage post-coiling recurrences and outline an approach to finding concrete, practical answers to common clinical questions.

Results

Although some maxims and general statements can be reluctantly offered, in the absence of evidence the best approach is to emphasize proper methods in order to one day find reliable answers. These can only come from randomized clinical trials. The multitude of research questions is an added difficulty. A program of necessary trials is proposed and centers are encouraged to work in a collaborative fashion in order to offer best possible care as well as progress.

Conclusion

Common clinical questions, including how to follow coiled aneurysm patients, if and when to re-treat, and with which treatment modality, are best addressed with the sound scientific methodology of clinical trials.
Pneumoconiosis and emphysema in construction workers: results of HRCT and lung function findings
Authors: Meijer E , Tjoe Nij E , Kraus T , van der Zee J S , van Delden O , van Leeuwen M , Lammers J W , Heederik D
Published: February 2011

Objectives

To evaluate the prevalence of HRCT findings in construction workers previously surveyed by chest radiographs classified according to ILO guidelines. To examine the association between HRCT findings and exposure to quartz containing dust, and lung function.

Methods

The study comprised a questionnaire, dynamic and static lung function measurements, single-breath CO diffusion capacity, chest radiographs and HRCT in 79 individuals. Certified ‘B’ readers coded radiographs according to the ILO classification. HRCT scans were read according to an international classification system. A qualitative exposure index for cumulative respiratory quartz on a 10-point scale was used.

Results

Agreement between HRCT readers was good (κ>0.60), except for irregular opacities (κ=0.23). In ILO category 0/0, 8% HRCT round, 22% irregular and/or linear opacities and 41% HRCT emphysema was found. HRCT round opacities was associated with high cumulative quartz exposure (OR 7.1; 95% CI 1.3 to 37.8). Emphysema was associated with smoking (OR 10.1; 95% CI 1.2 to 84.2) and showed a reduction in TL,CO,sb. HRCT round opacities was not associated with lung function. Current smoking was negatively associated with FEV1/FVC ratio and positively with RV/TLC ratio, and showed a reduction in TL,CO,sb(13.4%), adjusted for different HRCT findings.

Conclusions

Low grade silicosis cannot be excluded in workers with normal chest radiographs (ILO 0/0). In relatively highly exposed construction workers, a sevenfold increased risk of simple (nodular) silicosis was found. Emphysema on HRCT was associated with current or former smokers, but not with exposure, and contributed to reduced diffusion capacity. Airflow limitation was mainly determined by current smoking and was not associated with simple (nodular) silicosis.
Osteochondrosis lesions of the lateral trochlear ridge of the distal femur in four ponies
Authors: Voute L. C. , Henson F. M. D. , Platt D. , Jeffcott L. B.
Published: February 2011

Lesions of the lateral trochlear ridge (LTR) of the distal femur were investigated in four pony or pony cross horses. The animals were all geldings and were six to 15 months of age. Lesions were bilateral in three ponies and unilateral in one. Femoropatellar joint effusion and lameness were present in two ponies; clinical signs were absent in the others. The proximal LTR was affected in all four animals. The radiographic appearance of the lesions was a subchondral defect containing mineralised bodies. Arthroscopic and postmortem examination findings included an osteochondral flap, a fissured or irregular articular surface and a smooth surface overlying focally thickened cartilage that extended into subchondral bone. Thickened articular cartilage was a histological feature of all the lesions. Among the other histological features, the most common were chondronecrosis, chondrocyte clusters, phenotypically abnormal chondrocytes, horizontal fissures at the osteochondral junction and retained blood vessels. The signalment of the four ponies, their clinical signs and the pathological features of their lesions were consistent with osteochondrosis of the LTR in horses. The use of multiple criteria was considered to be important in making a specific diagnosis.
Impact of surgical ventricular restoration on ventricular shape, wall stress, and function in heart failure patients
Authors: Zhong L. , Su Y. , Gobeawan L. , Sola S. , Tan R.-S. , Navia J. L. , Ghista D. N. , Chua T. , Guccione J. , Kassab G. S.
Published: February 2011

Surgical ventricular restoration (SVR) was designed to treat patients with aneurysms or large akinetic walls and dilated ventricles. Yet, crucial aspects essential to the efficacy of this procedure like optimal shape and size of the left ventricle (LV) are still debatable. The objective of this study is to quantify the efficacy of SVR based on LV regional shape in terms of curvedness, wall stress, and ventricular systolic function. A total of 40 patients underwent magnetic resonance imaging (MRI) before and after SVR. Both short-axis and long-axis MRI were used to reconstruct end-diastolic and end-systolic three-dimensional LV geometry. The regional shape in terms of surface curvedness, wall thickness, and wall stress indexes were determined for the entire LV. The infarct, border, and remote zones were defined in terms of end-diastolic wall thickness. The LV global systolic function in terms of global ejection fraction, the ratio between stroke work (SW) and end-diastolic volume (SW/EDV), the maximal rate of change of pressure-normalized stress (dσ*/d tmax), and the regional function in terms of surface area change were examined. The LV end-diastolic and end-systolic volumes were significantly reduced, and global systolic function was improved in ejection fraction, SW/EDV, and dσ*/d tmax. In addition, the end-diastolic and end-systolic stresses in all zones were reduced. Although there was a slight increase in regional curvedness and surface area change in each zone, the change was not significant. Also, while SVR reduced LV wall stress with increased global LV systolic function, regional LV shape and function did not significantly improve.
p38 MAPK-dependent small HSP27 and αB-crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest
Authors: Clements Richard T. , Feng Jun , Cordeiro Brenda , Bianchi Cesario , Sellke Frank W.
Published: February 2011

We previously demonstrated that myocardial p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) are phosphorylated following cardioplegic arrest in patients undergoing cardiac surgery and correlate with reduced cardiac function. The following studies were performed to determine whether inhibition of p38 MAPK and/or overexpression of nonphosphorylatable HSP27 improves cardiac function following cardioplegic arrest. Langendorff-perfused isolated rat hearts were subjected to 2 h of intermittent cold cardioplegia followed by 30 min of reperfusion. Hearts were treated with (CP+SB) or without (CP) the p38 MAPK inhibitor SB-203580 (5 μM) supplied in the cardioplegia. Sham-treated hearts served as controls. In separate experiments, isolated rat ventricular myocytes infected with either green fluorescent protein (GFP) or a nonphosphorylatable HSP27 mutant (3A-HSP27) were subjected to 3 h of cold hypoxic cardioplegia and simulated reperfusion (CP) followed by video microscopy and length change measurements. Baseline parameters of cardiac function were similar between groups [left ventricular developed pressure (LVDP), 119 ± 4.9 mmHg; positive and negative first derivatives of LV pressure (± dP/d t), 3,139 ± 245 and 2, 314 ± 110 mmHg/s]. CP resulted in reduced cardiac function (LVDP, 72.2 ± 5.8 mmHg; ± dP/d t, 2,076 ± 231 and −1,317 ± 156 mmHg/s) compared with baseline. Treatment with 5 μM SB-203580 significantly improved CP-induced cardiac function (LVDP, 101.9 ± 0 mmHg; ±dP/d t, 2,836 ± 163 and −2,108 ± 120 mmHg/s; P = 0.03, 0.01, and 0.04, CP+SB vs. CP). Inhibition of p38 MAPK significantly lowered CP-induced p38 MAPK, HSP27, and αB-crystallin (cryAB) phosphorylation. In vitro CP decreased myocyte length changes from 10.3 ± 1.5% (GFP) to 5.7 ± 0.8% (GFP+CP). Infection with 3A-HSP27 completely rescued CP-induced decreased myocyte contraction (11.1 ± 1.0%). However, infection with 3A-HSP27 did not block the endogenous HSP27 response. We conclude that inhibition of p38 MAPK and subsequent HSP27 and cryAB phosphorylation and/or overexpression of nonphosphorylatable HSP27 significantly improves cardiac performance following cardioplegic arrest. Modulation of HSP27 phosphorylation may improve myocardial stunning following cardiac surgery.
Rapamycin treatment augments both protein ubiquitination and Akt activation in pressure-overloaded rat myocardium
Authors: Harston Rebecca K. , McKillop John C. , Moschella Phillip C. , Van Laer An , Quinones Lakeya S. , Baicu Catalin F. , Balasubramanian Sundaravadivel , Zile Michael R. , Kuppuswamy Dhandapani
Published: February 2011

Ubiquitin-mediated protein degradation is necessary for both increased ventricular mass and survival signaling for compensated hypertrophy in pressure-overloaded (PO) myocardium. Another molecular keystone involved in the hypertrophic growth process is the mammalian target of rapamycin (mTOR), which forms two distinct functional complexes: mTORC1 that activates p70S6 kinase-1 to enhance protein synthesis and mTORC2 that activates Akt to promote cell survival. Independent studies in animal models show that rapamycin treatment that alters mTOR complexes also reduces hypertrophic growth and increases lifespan by an unknown mechanism. We tested whether the ubiquitin-mediated regulation of growth and survival in hypertrophic myocardium is linked to the mTOR pathway. For in vivo studies, right ventricle PO in rats was conducted by pulmonary artery banding; the normally loaded left ventricle served as an internal control. Rapamycin (0.75 mg/kg per day) or vehicle alone was administered intraperitoneally for 3 days or 2 wk. Immunoblot and immunofluorescence imaging showed that the level of ubiquitylated proteins in cardiomyocytes that increased following 48 h of PO was enhanced by rapamycin. Rapamycin pretreatment also significantly increased PO-induced Akt phosphorylation at S473, a finding confirmed in cardiomyocytes in vitro to be downstream of mTORC2. Analysis of prosurvival signaling in vivo showed that rapamycin increased PO-induced degradation of phosphorylated inhibitor of κB, enhanced expression of cellular inhibitor of apoptosis protein 1, and decreased active caspase-3. Long-term rapamycin treatment in 2-wk PO myocardium blunted hypertrophy, improved contractile function, and reduced caspase-3 and calpain activation. These data indicate potential cardioprotective benefits of rapamycin in PO hypertrophy.
Attenuation of sympathetic baroreflex sensitivity during the onset of acute mental stress in humans
Authors: Durocher John J. , Klein Jenna C. , Carter Jason R.
Published: February 2011

Mental stress consistently induces a pressor response that is often accompanied by a paradoxical increase of muscle sympathetic nerve activity (MSNA). The purpose of the present study was to evaluate sympathetic baroreflex sensitivity (BRS) by examining the relations between spontaneous fluctuations of diastolic arterial pressure (DAP) and MSNA. We hypothesized that sympathetic BRS would be attenuated during mental stress. DAP and MSNA were recorded during 5 min of supine baseline, 5 min of mental stress, and 5 min of recovery in 32 young healthy adults. Burst incidence and area were determined for each cardiac cycle and placed into 3-mmHg DAP bins; the slopes between DAP and MSNA provided an index of sympathetic BRS. Correlations between DAP and MSNA were strong (>0.5) during baseline in 31 of 32 subjects, but we evaluated the change in slope only for those subjects maintaining a strong correlation during mental stress (16 subjects). During baseline, the relation between DAP and MSNA was negative when expressed as either burst incidence [slope = −1.95 ± 0.18 bursts·(100 beats)−1·mmHg−1; r = −0.86 ± 0.03] or total MSNA [slope = −438 ± 91 units·(beat)−1 mmHg−1; r = −0.76 ± 0.06]. During mental stress, the slope between burst incidence and DAP was significantly reduced [slope = −1.14 ± 0.12 bursts·(100 beats)−1·mmHg−1; r = −0.72 ± 0.03; P < 0.01], indicating attenuation of sympathetic BRS. A more detailed analysis revealed an attenuation of sympathetic BRS during the first 2 min of mental stress ( P < 0.01) but no change during the final 3 min of mental stress ( P = 0.25). The present study demonstrates that acute mental stress attenuates sympathetic BRS, which may partially contribute to sympathoexcitation during the mental stress-pressor response. However, this attenuation appears to be isolated to the onset of mental stress. Moreover, variable MSNA responses to mental stress do not appear to be directly related to sympathetic BRS.
Mild DOCA-salt hypertension: sympathetic system and role of renal nerves
Authors: Kandlikar Sachin S. , Fink Gregory D.
Published: February 2011

Excess sympathetic nervous system activity (SNA) is linked to human essential and experimental hypertension. To test whether sympathetic activation is associated with a model of deoxycorticosterone acetate (DOCA)-salt hypertension featuring two kidneys and a moderate elevation of blood pressure, we measured whole body norepinephrine (NE) spillover as an index of global SNA. Studies were conducted in chronically catheterized male Sprague-Dawley rats drinking water containing 1% NaCl and 0.2% KCl. After a 7-day surgical recovery and a 3-day control period, a DOCA pellet (50 mg/kg) was implanted subcutaneously in one group of rats (DOCA), while the other group underwent sham implantation (Sham). NE spillover was measured on control day 2 and days 7 and 14 after DOCA administration or sham implantation. During the control period, mean arterial pressure (MAP) was similar in Sham and DOCA rats. MAP was significantly increased in the DOCA group compared with the Sham group after DOCA administration ( day 14: Sham = 109 ± 5.3, DOCA = 128 ± 3.6 mmHg). However, plasma NE concentration, clearance, and spillover were not different in the two groups at any time. To determine whether selective sympathetic activation to the kidneys contributes to hypertension development, additional studies were performed in renal denervated (RDX) and sham-denervated (Sham-DX) rats. MAP, measured by radiotelemetry, was similar in both groups during the control and DOCA treatment periods. In conclusion, global SNA is not increased during the development of mild DOCA-salt hypertension, and fully intact renal nerves are not essential for hypertension development in this model.
A mathematical model of spontaneous calcium release in cardiac myocytes
Authors: Chen Wei , Aistrup Gary , Wasserstrom J. Andrew , Shiferaw Yohannes
Published: February 2011

In cardiac myocytes, calcium (Ca) can be released from the sarcoplasmic reticulum independently of Ca influx from voltage-dependent membrane channels. This efflux of Ca, referred to as spontaneous Ca release (SCR), is due to Ryanodine receptor fluctuations, which can induce spontaneous Ca sparks, which propagate to form Ca waves. This release of Ca can then induce delayed after-depolarizations (DADs), which can lead to arrhythmogenic-triggered activity in the heart. However, despite its importance, to date there is no mathematical model of SCR that accounts for experimentally observed features of subcellular Ca. In this article, we present an experimentally based model of SCR that reproduces the timing distribution of spontaneous Ca sparks and key features of the propagation of Ca waves emanating from these spontaneous sparks. We have coupled this model to an ionic model for the rabbit ventricular action potential to simulate SCR within several thousand cells in cardiac tissue. We implement this model to study the formation of an ectopic beat on a cable of cells that exhibit SCR-induced DADs.
PPARγ agonist rosiglitazone prevents perinatal nicotine exposure-induced asthma in rat offspring
Authors: Liu Jie , Sakurai Reiko , O'Roark E. M. , Kenyon Nicholas J. , Torday John S. , Rehan Virender K.
Published: February 2011

Perinatal exposure to maternal smoke is associated with adverse pulmonary effects, including reduced lung function and increased incidence of asthma. However, the mechanisms underlying these effects are unknown, and there is no effective preventive and/or therapeutic intervention. Recently, we suggested that downregulation of homeostatic mesenchymal peroxisome proliferator-activated receptor-γ (PPARγ) signaling following in utero nicotine exposure might contribute to chronic lung diseases such as asthma. We used an in vivo rat model to determine the effect of perinatal nicotine exposure on 1) offspring pulmonary function, 2) mesenchymal markers of airway contractility in trachea and lung tissue, and 3) whether administration of a PPARγ agonist, rosiglitazone (RGZ), blocks the molecular and functional effects of perinatal nicotine exposure on offspring lung. Pregnant Sprague-Dawley rat dams received placebo, nicotine, or nicotine + RGZ daily from embryonic day 6 until postnatal day 21, when respiratory system resistance, compliance, tracheal contractility, and the expression of markers of pulmonary contractility were determined. A significant increase in resistance and a decrease in compliance under basal conditions, with more pronounced changes following methacholine challenge, were observed with perinatal nicotine exposure compared with control. Tracheal constriction response and expression of mesenchymal markers of airway contractility were also significantly increased following perinatal nicotine exposure. Concomitant treatment with RGZ completely blocked the nicotine-induced alterations in pulmonary function, as well as the markers of airway contractility, at proximal and distal airway levels. These data suggest that perinatal smoke exposure-induced asthma can be effectively blocked by PPARγ agonists.
Identification of a Soluble Form of B7-H1 That Retains Immunosuppressive Activity and Is Associated with Aggressive Renal Cell Carcinoma
Authors: Frigola Xavier , Inman Brant A. , Lohse Christine M. , Krco Christopher J. , Cheville John C. , Thompson R. Houston , Leibovich Bradley , Blute Michael L. , Dong Haidong , Kwon Eugene D.
Published: February 2011

Purpose: Release of inhibitory coregulatory proteins into the circulation may represent one mechanism by which tumors thwart immune responses. Our objective was to determine whether soluble B7-H1 (sB7-H1) levels in patients with clear cell renal cell carcinoma (ccRCC) are associated with pathologic features and patient outcome.

Experimental Design: We developed an ELISA for quantification of sB7-H1 in biological fluids. Biochemical confirmation of the measured analyte as sB7-H1 was done by protein microsequencing using supernates from tumor cell lines. Biological activity of sB7-H1 was assessed in vitro utilizing T-cell apoptosis assays. We tested sB7-H1 levels in the sera from 172 ccRCC patients and correlated sB7-H1 levels with pathologic features and patient outcome.

Results: sB7-H1 was detected in the cell supernatants of some B7-H1–positive tumor cell lines. Protein sequencing established that the measured sB7-H1 retained its receptor-binding domain and could deliver proapoptotic signals to T cells. Higher preoperative sB7-H1 levels were associated with larger tumors (P < 0.001), tumors of advanced stage (P = 0.017) and grade (P = 0.044), and tumors with necrosis (P = 0.003). A doubling of sB7-H1 levels was associated with a 41% increased risk of death (P = 0.010).

Conclusion: Our observations suggest that sB7-H1 may be detected in the sera of ccRCC patients and that sB7-H1 may systemically impair host immunity, thereby fostering cancer progression and subsequent poor clinical outcome. Clin Cancer Res; 17(7); 1915–23. ©2011 AACR.
Comparative Prognostic Value of Epidermal Growth Factor Quantitative Protein Expression Compared with FISH for Head and Neck Squamous Cell Carcinoma
Authors: Pectasides Eirini , Rampias Theodore , Kountourakis Panteleimon , Sasaki Clarence , Kowalski Diane , Fountzilas George , Zaramboukas Thomas , Rimm David , Burtness Barbara , Psyrri Amanda
Published: February 2011

Purpose: Epidermal growth factor receptor (EGFR) overexpression correlates with recurrence and with treatment resistance in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to evaluate the relationship of EGFR gene copy number utilizing FISH and protein expression with automated quantitative analysis (AQUA) and to correlate those with patient outcome.

Experimental Design: A tissue microarray composed of 102 HNSCC treated with (chemo)radiation was constructed and analyzed for EGFR copy number by FISH (Vysis; Abbott Laboratories) and EGFR protein expression using AQUA analysis of EGFR staining scored on a scale of 0 to 255. We evaluated associations of EGFR FISH status and AQUA score with clinicopathologic parameters and survival prognosis.

Results: Eleven (17.2%) of 64 tumors with FISH results showed EGFR high polysomy and/or gene amplification (FISH positive). Protein levels assessed by AQUA in FISH-positive cases were significantly higher (P = 0.04) than in FISH-negative cases. Using the continuous AQUA scores for EGFR expression, AQUA and FISH showed significant agreement (Pearson's ρ = 0.353, P = 0.04). Patients with high tumor EGFR protein expression had inferior 5-year overall survival (27.7%) compared with those with low tumor EGFR expression (54%; P = 0.029). There was no significant association between EGFR FISH status and overall survival (P = 0.201). In the multivariate model, high tumor EGFR protein expression status remained an independent prognostic factor for overall survival (P = 0.047).

Conclusions: EGFR protein content correlates with gene copy number if protein content is quantitated and automatically analyzed, as with AQUA. EGFR protein levels assessed by AQUA strongly predict for patient outcome in HNSCC, whereas EGFR FISH status does not provide prognostic information. Clin Cancer Res; 17(9); 2947–54. ©2011 AACR.
Carfilzomib-Dependent Selective Inhibition of the Chymotrypsin-like Activity of the Proteasome Leads to Antitumor Activity in Waldenstrom's Macroglobulinemia
Authors: Sacco Antonio , Aujay Monette , Morgan Brittany , Azab Abdel Kareem , Maiso Patricia , Liu Yang , Zhang Yong , Azab Feda , Ngo Hai T. , Issa Ghayas C. , Quang Phong , Roccaro Aldo M. , Ghobrial Irene M.
Published: February 2011

Purpose: Primary Waldenstrom's Macroglobulinemia (WM) cells present with a significantly higher level of the immunoproteasome compared with the constitutive proteasome. It has been demonstrated that selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive-(c20S) and immuno-(i20S) proteasome represents a valid strategy to induce antineoplastic effect in hematologic tumors. We therefore evaluated carfilzomib, a potent selective, irreversible inhibitor of the CT-L activity of the i20S and c20S in WM cells.

Experimental Design: We tested the effect of carfilzomib on survival and proliferation of primary WM cells, as well as of other IgM-secreting lymphoma cell lines. Carfilzomib-dependent mechanisms of induced apoptosis in WM cells, and its effect on WM cells in the context of bone marrow (BM) microenvironment have been also evaluated. Moreover, the combinatory effect of carfilzomib and bortezomib has been investigated. In vivo studies have been performed.

Results: We demonstrated that carfilzomib targeted the CT-L activity of both i20S and c20S, which led to the induction of toxicity in primary WM cells, as well as in other IgM-secreting lymphoma cells. Importantly, carfilzomib targeted WM cells even in the context of BM milieu. In addition, carfilzomib induced apoptosis through c-jun-N-terminal-kinase activation, caspase cleavage, and initiation of unfolded protein response. Importantly, the combination of carfilzomib and bortezomib synergistically inhibited CT-L activity, as well as caspase-, PARP-cleavage and GRP94 expression. Antitumor activity of carfilzomib has been validated in vivo.

Conclusions: These findings suggest that targeting i20S and c20S CT-L activity by carfilzomib represents a valid antitumor strategy in WM and other IgM-secreting lymphomas. Clin Cancer Res; 17(7); 1753–64. ©2011 AACR.
Human Prostate Cancer Harbors the Stem Cell Properties of Bone Marrow Mesenchymal Stem Cells
Authors: Zhau Haiyen E. , He Hui , Wang Christopher Y. , Zayzafoon Majd , Morrissey Colm , Vessella Robert L. , Marshall Fray F. , Chung Leland W. K. , Wang Ruoxiang
Published: February 2011

Purpose: Prostate tumor cells frequently show the features of osteoblasts, which are differentiated from bone marrow mesenchymal stem cells. We examined human prostate cancer cell lines and clinical prostate cancer specimens for additional bone marrow mesenchymal stem cell properties.

Experimental Design: Prostate cancer cell lines were induced for osteoblastogenic and adipogenic differentiation, detected by standard staining methods and confirmed by lineage-specific marker expression. Abnormal expression of the markers was then assessed in clinical prostate cancer specimens.

Results: After osteoblastogenic induction, cells of the LNCaP lineage, PC-3 lineage, and DU145 displayed osteoblastic features. Upon adipogenic induction, PC-3 lineage and DU145 cells differentiated into adipocyte-like cells. The adipocyte-like cancer cells expressed brown adipocyte-specific markers, suggesting differentiation along the brown adipocyte lineage. The adipogenic differentiation was accompanied by growth inhibition, and most of the adipocyte-like cancer cells were committed to apoptotic death. During cyclic treatments with adipogenic differentiation medium and then with control medium, the cancer cells could commit to repeated adipogenic differentiation and retrodifferentiation. In clinical prostate cancer specimens, the expression of uncoupling protein 1 (UCP1), a brown fat-specific marker, was enhanced with the level of expression correlated to disease progression from primary to bone metastatic cancers.

Conclusions: This study thus revealed that prostate cancer cells harbor the stem cell properties of bone marrow mesenchymal stem cells. The abnormally expressed adipogenic UCP1 protein may serve as a unique marker, while adipogenic induction can be explored as a differentiation therapy for prostate cancer progression and bone metastasis. Clin Cancer Res; 17(8); 2159–69. ©2011 AACR.
Targeting Aberrant PI3K/Akt Activation by PI103 Restores Sensitivity to TRAIL-Induced Apoptosis in Neuroblastoma
Authors: Opel Daniela , Naumann Ivonne , Schneider Maxi , Bertele Daniela , Debatin Klaus-Michael , Fulda Simone
Published: February 2011

Purpose: Because we recently identified Akt activation as a novel poor prognostic indicator in neuroblastoma, we investigated whether phosphoinositide 3′-kinase (PI3K) inhibition sensitizes neuroblastoma cells for TRAIL-induced apoptosis.

Experimental Design: The effect of pharmacological or genetic inhibition of PI3K or mTOR was analyzed on apoptosis induction, clonogenic survival, and activation of apoptosis signaling pathways in vitro and in a neuroblastoma in vivo model. The functional relevance of individual Bcl-2 family proteins was examined by knockdown or overexpression experiments.

Results: The PI3K inhibitor PI103 cooperates with TRAIL to synergistically induce apoptosis (combination index < 0.1), to suppress clonogenic survival, and to reduce tumor growth in a neuroblastoma in vivo model. Similarly, genetic silencing of PI3K significantly increases TRAIL-mediated apoptosis, whereas genetic or pharmacological blockage of mTOR fails to potentiate TRAIL-induced apoptosis. Combined treatment with PI103 and TRAIL enhances cleavage of Bid and the insertion of tBid into mitochondrial membranes, and reduces phosphorylation of BimEL. Additionally, PI103 decreases expression of Mcl-1, XIAP, and cFLIP, thereby promoting Bax/Bak activation, mitochondrial perturbations, and caspase-dependent apoptosis. Knockdown of Bid or Noxa or overexpression of Bcl-2 rescues cells from PI103- and TRAIL-induced apoptosis, whereas Mcl-1 silencing potentiates apoptosis. Bcl-2 overexpression also inhibits cleavage of caspase-3, caspase-8, and Bid pointing to a mitochondria-driven feedback amplification loop.

Conclusions: PI103 primes neuroblastoma cells for TRAIL-induced apoptosis by shifting the balance toward proapoptotic Bcl-2 family members and increased mitochondrial apoptosis. Thus, PI3K inhibitors represent a novel promising approach to enhance the efficacy of TRAIL-based treatment protocols in neuroblastoma. Clin Cancer Res; 17(10); 3233–47. ©2011 AACR.
DNA Damage Persistence as Determinant of Tumor Sensitivity to the Combination of Topo I Inhibitors and Telomere-Targeting Agents
Authors: Biroccio Annamaria , Porru Manuela , Rizzo Angela , Salvati Erica , D'Angelo Carmen , Orlandi Augusto , Passeri Daniela , Franceschin Marco , Stevens Malcolm F.G. , Gilson Eric , Beretta Giovanni , Zupi Gabriella , Pisano Claudio , Zunino Franco , Leonetti Carlo
Published: February 2011

Purpose: We previously reported that the G-quadruplex (G4) ligand RHPS4 potentiates the antitumor activity of camptothecins both in vitro and in tumor xenografts. The present study aims at investigating the mechanisms involved in this specific drug interaction.

Experimental Design: Combination index test was used to evaluate the interaction between G4 ligands and standard or novel Topo I inhibitors. Chromatin immunoprecipitation was performed to study the presence at telomeres of various types of topisomerase, while immunolabeling experiments were performed to measure the activation of DNA damage both in vitro and in tumor xenografts.

Results: We report that integration of the Topo I inhibitor SN-38, but not the Topo II poison doxorubicin with telomere-based therapy is strongly effective and the sequence of drug administration is critical in determining the synergistic interaction, impairing the cell ability to recover from drug-induced cytotoxicity. The synergistic effect of this combination was also observed by using novel camptothecins and, more interestingly, mice treated with ST1481/RHPS4 combination showed an inhibition and delay of tumor growth as well as an increased survival. The study of the mechanism(s) revealed that treatment with G4 ligands increased Topo I at the telomeres and the functional relevance of this observation was directly assessed by showing that standard and novel camptothecins stabilized DNA damage both in vitro and in xenografts.

Conclusions: Our results demonstrate an outstanding efficacy of Topo I inhibitors/G4 ligands combination, which likely reflects an enhanced and persistent activation of DNA damage response as a critical determinant of the therapeutic improvement. Clin Cancer Res; 17(8); 2227–36. ©2011 AACR.