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1.  A risk prediction algorithm based on family history and common genetic variants: application to prostate cancer with potential clinical impact 
Genetic epidemiology  2011;35(6):549-556.
Genome Wide Association Studies have identified several Single Nucleotide Polymorphisms (SNPs) that are independently associated with small increments in risk of prostate cancer, opening up the possibility for using such variants in risk prediction. Using segregation analysis of population-based samples of 4390 families of prostate cancer patients from the UK and Australia, and assuming all familial aggregation has genetic causes, we previously found that the best model for the genetic susceptibility to prostate cancer was a mixed model of inheritance that included both a recessive major gene component and a polygenic component (P) that represents the effect of a large number of genetic variants each of small effect, where P∼N(0,σP2). Based on published studies of 26 SNPs that are currently known to be associated with prostate cancer, we have extended our model to incorporate these SNPs by decomposing the polygenic component into two parts: a polygenic component due to the known susceptibility SNPs, PK∼N(0,σK2), and the residual polygenic component due to the postulated but as yet unknown genetic variants, PU∼N(0,σU2). The resulting algorithm can be used for predicting the probability of developing prostate cancer in the future based on both SNP profiles and explicit family history information. This approach can be applied to other diseases for which population-based family data and established risk variants exist.
PMCID: PMC3950816  PMID: 21769933
2.  A meta-analysis of genome-wide association studies to identify prostate cancer susceptibility loci associated with aggressive and non-aggressive disease 
Amin Al Olama, Ali | Kote-Jarai, Zsofia | Schumacher, Fredrick R. | Wiklund, Fredrik | Berndt, Sonja I. | Benlloch, Sara | Giles, Graham G. | Severi, Gianluca | Neal, David E. | Hamdy, Freddie C. | Donovan, Jenny L. | Hunter, David J. | Henderson, Brian E. | Thun, Michael J. | Gaziano, Michael | Giovannucci, Edward L. | Siddiq, Afshan | Travis, Ruth C. | Cox, David G. | Canzian, Federico | Riboli, Elio | Key, Timothy J. | Andriole, Gerald | Albanes, Demetrius | Hayes, Richard B. | Schleutker, Johanna | Auvinen, Anssi | Tammela, Teuvo L.J. | Weischer, Maren | Stanford, Janet L. | Ostrander, Elaine A. | Cybulski, Cezary | Lubinski, Jan | Thibodeau, Stephen N. | Schaid, Daniel J. | Sorensen, Karina D. | Batra, Jyotsna | Clements, Judith A. | Chambers, Suzanne | Aitken, Joanne | Gardiner, Robert A. | Maier, Christiane | Vogel, Walther | Dörk, Thilo | Brenner, Hermann | Habuchi, Tomonori | Ingles, Sue | John, Esther M. | Dickinson, Joanne L. | Cannon-Albright, Lisa | Teixeira, Manuel R. | Kaneva, Radka | Zhang, Hong-Wei | Lu, Yong-Jie | Park, Jong Y. | Cooney, Kathleen A. | Muir, Kenneth R. | Leongamornlert, Daniel A. | Saunders, Edward | Tymrakiewicz, Malgorzata | Mahmud, Nadiya | Guy, Michelle | Govindasami, Koveela | O'Brien, Lynne T. | Wilkinson, Rosemary A. | Hall, Amanda L. | Sawyer, Emma J. | Dadaev, Tokhir | Morrison, Jonathan | Dearnaley, David P. | Horwich, Alan | Huddart, Robert A. | Khoo, Vincent S. | Parker, Christopher C. | Van As, Nicholas | Woodhouse, Christopher J. | Thompson, Alan | Dudderidge, Tim | Ogden, Chris | Cooper, Colin S. | Lophatonanon, Artitaya | Southey, Melissa C. | Hopper, John L. | English, Dallas | Virtamo, Jarmo | Le Marchand, Loic | Campa, Daniele | Kaaks, Rudolf | Lindstrom, Sara | Diver, W. Ryan | Gapstur, Susan | Yeager, Meredith | Cox, Angela | Stern, Mariana C. | Corral, Roman | Aly, Markus | Isaacs, William | Adolfsson, Jan | Xu, Jianfeng | Zheng, S. Lilly | Wahlfors, Tiina | Taari, Kimmo | Kujala, Paula | Klarskov, Peter | Nordestgaard, Børge G. | Røder, M. Andreas | Frikke-Schmidt, Ruth | Bojesen, Stig E. | FitzGerald, Liesel M. | Kolb, Suzanne | Kwon, Erika M. | Karyadi, Danielle M. | Orntoft, Torben Falck | Borre, Michael | Rinckleb, Antje | Luedeke, Manuel | Herkommer, Kathleen | Meyer, Andreas | Serth, Jürgen | Marthick, James R. | Patterson, Briony | Wokolorczyk, Dominika | Spurdle, Amanda | Lose, Felicity | McDonnell, Shannon K. | Joshi, Amit D. | Shahabi, Ahva | Pinto, Pedro | Santos, Joana | Ray, Ana | Sellers, Thomas A. | Lin, Hui-Yi | Stephenson, Robert A. | Teerlink, Craig | Muller, Heiko | Rothenbacher, Dietrich | Tsuchiya, Norihiko | Narita, Shintaro | Cao, Guang-Wen | Slavov, Chavdar | Mitev, Vanio | Chanock, Stephen | Gronberg, Henrik | Haiman, Christopher A. | Kraft, Peter | Easton, Douglas F. | Eeles, Rosalind A.
Human Molecular Genetics  2012;22(2):408-415.
Genome-wide association studies (GWAS) have identified multiple common genetic variants associated with an increased risk of prostate cancer (PrCa), but these explain less than one-third of the heritability. To identify further susceptibility alleles, we conducted a meta-analysis of four GWAS including 5953 cases of aggressive PrCa and 11 463 controls (men without PrCa). We computed association tests for approximately 2.6 million SNPs and followed up the most significant SNPs by genotyping 49 121 samples in 29 studies through the international PRACTICAL and BPC3 consortia. We not only confirmed the association of a PrCa susceptibility locus, rs11672691 on chromosome 19, but also showed an association with aggressive PrCa [odds ratio = 1.12 (95% confidence interval 1.03–1.21), P = 1.4 × 10−8]. This report describes a genetic variant which is associated with aggressive PrCa, which is a type of PrCa associated with a poorer prognosis.
PMCID: PMC3526158  PMID: 23065704
3.  Long-term functional donor site morbidity of the free radial forearm flap in head and neck cancer survivors 
To assess the functional donor site morbidity of the forearm free flap in patients surviving at least 2 years after ablative head and neck cancer surgery in a tertiary care centre.
This study involved nine long-term survivors (2 year post-operative) who had forearm free flaps to reconstruct head and neck defects. All flaps were raised from the non-dominant arm. The non-donor side acted as a control for all patients. Objective measurements were as follows: grip, tip pinch and key pinch strength measured with dynamometers; flexion, extension, radial and ulnar deviation and pronation and supination range of motion at the wrist measured with goniometry; A timed manual dexterity task was performed with a grooved pegboard test, and sensation of the radial nerve was tested with Semmes Weinstein monofilaments. Subjective measurements included a validated patient questionnaire of hand function and opinions of scar appearance as well as a validated scar assessment from two different observers.
Pronation at the wrist, manual dexterity and sensation were found to be significantly reduced in the donor side compared to the non-donor side. Inter-rater agreement between the two observers was found to be poor, except for an acceptable correlation between overall scar opinions. No correlations were found between any subjective or objective items or between the patient’s and the observers’ subjective evaluations.
Donor site morbidity can be demonstrated with objective testing however this is accepted and well tolerated by head and neck cancer patients.
PMCID: PMC3895707  PMID: 24418459
Radial forearm flap; Donor-site; Morbidity; Long-term
4.  Heat Shock Transcription Factor σ32 Co-opts the Signal Recognition Particle to Regulate Protein Homeostasis in E. coli 
PLoS Biology  2013;11(12):e1001735.
The bacterial heat shock transcription factor, σ32, maintains proper protein homeostasis only after it is targeted to the inner membrane by the signal recognition particle (SRP), thereby enabling integration of protein folding information from both the cytoplasm and cell membrane.
All cells must adapt to rapidly changing conditions. The heat shock response (HSR) is an intracellular signaling pathway that maintains proteostasis (protein folding homeostasis), a process critical for survival in all organisms exposed to heat stress or other conditions that alter the folding of the proteome. Yet despite decades of study, the circuitry described for responding to altered protein status in the best-studied bacterium, E. coli, does not faithfully recapitulate the range of cellular responses in response to this stress. Here, we report the discovery of the missing link. Surprisingly, we found that σ32, the central transcription factor driving the HSR, must be localized to the membrane rather than dispersed in the cytoplasm as previously assumed. Genetic analyses indicate that σ32 localization results from a protein targeting reaction facilitated by the signal recognition particle (SRP) and its receptor (SR), which together comprise a conserved protein targeting machine and mediate the cotranslational targeting of inner membrane proteins to the membrane. SRP interacts with σ32 directly and transports it to the inner membrane. Our results show that σ32 must be membrane-associated to be properly regulated in response to the protein folding status in the cell, explaining how the HSR integrates information from both the cytoplasm and bacterial cell membrane.
Author Summary
All cells have to adjust to frequent changes in their environmental conditions. The heat shock response is a signaling pathway critical for survival of all organisms exposed to elevated temperatures. Under such conditions, the heat shock response maintains enzymes and other proteins in a properly folded state. The mechanisms for sensing temperature and the subsequent induction of the appropriate transcriptional response have been extensively studied. Prior to this work, however, the circuitry described in the best studied bacterium E. coli could not fully explain the range of cellular responses that are observed following heat shock. We report the discovery of this missing link. Surprisingly, we find that σ32, a transcription factor that induces gene expression during heat shock, needs to be localized to the membrane, rather than being active as a soluble cytoplasmic protein as previously thought. We show that, equally surprisingly, σ32 is targeted to the membrane by the signal recognition particle (SRP) and its receptor (SR). SRP and SR constitute a conserved protein targeting machine that normally only operates on membrane and periplasmic proteins that contain identifiable signal sequences. Intriguingly, σ32 does not have any canonical signal sequence for export or membrane-integration. Our results indicate that membrane-associated σ32, not soluble cytoplasmic σ32, is the preferred target of regulatory control in response to heat shock. Our new model thus explains how protein folding status from both the cytoplasm and bacterial cell membrane can be integrated to control the heat shock response.
PMCID: PMC3866087  PMID: 24358019
5.  Identification of 23 new prostate cancer susceptibility loci using the iCOGS custom genotyping array 
Eeles, Rosalind A | Olama, Ali Amin Al | Benlloch, Sara | Saunders, Edward J | Leongamornlert, Daniel A | Tymrakiewicz, Malgorzata | Ghoussaini, Maya | Luccarini, Craig | Dennis, Joe | Jugurnauth-Little, Sarah | Dadaev, Tokhir | Neal, David E | Hamdy, Freddie C | Donovan, Jenny L | Muir, Ken | Giles, Graham G | Severi, Gianluca | Wiklund, Fredrik | Gronberg, Henrik | Haiman, Christopher A | Schumacher, Fredrick | Henderson, Brian | Le Marchand, Loic | Lindstrom, Sara | Kraft, Peter | Hunter, David J | Gapstur, Susan | Chanock, Stephen J | Berndt, Sonja I | Albanes, Demetrius | Andriole, Gerald | Schleutker, Johanna | Weischer, Maren | Canzian, Federico | Riboli, Elio | Key, Tim J | Travis, Ruth | Campa, Daniele | Ingles, Sue A | John, Esther M | Hayes, Richard B | Pharoah, Paul DP | Pashayan, Nora | Khaw, Kay-Tee | Stanford, Janet | Ostrander, Elaine A | Signorello, Lisa B | Thibodeau, Stephen N | Schaid, Dan | Maier, Christiane | Vogel, Walther | Kibel, Adam S | Cybulski, Cezary | Lubinski, Jan | Cannon-Albright,  | Brenner, Hermann | Park, Jong Y | Kaneva, Radka | Batra, Jyotsna | Spurdle, Amanda B | Clements, Judith A | Teixeira, Manuel R | Dicks, Ed | Lee, Andrew | Dunning, Alison | Baynes, Caroline | Conroy, Don | Maranian, Melanie J | Ahmed, Shahana | Govindasami, Koveela | Guy, Michelle | Wilkinson, Rosemary A | Sawyer, Emma J | Morgan, Angela | Dearnaley, David P | Horwich, Alan | Huddart, Robert A | Khoo, Vincent S | Parker, Christopher C | Van As, Nicholas J | Woodhouse, J | Thompson, Alan | Dudderidge, Tim | Ogden, Chris | Cooper, Colin | Lophatananon, Artitaya | Cox, Angela | Southey, Melissa | Hopper, John L | English, Dallas R | Aly, Markus | Adolfsson, Jan | Xu, Jiangfeng | Zheng, Siqun | Yeager, Meredith | Kaaks, Rudolf | Diver, W Ryan | Gaudet, Mia M | Stern, Mariana | Corral, Roman | Joshi, Amit D | Shahabi, Ahva | Wahlfors, Tiina | Tammela, Teuvo J | Auvinen, Anssi | Virtamo, Jarmo | Klarskov, Peter | Nordestgaard, Børge G | Røder, Andreas | Nielsen, Sune F | Bojesen, Stig E | Siddiq, Afshan | FitzGerald, Liesel | Kolb, Suzanne | Kwon, Erika | Karyadi, Danielle | Blot, William J | Zheng, Wei | Cai, Qiuyin | McDonnell, Shannon K | Rinckleb, Antje | Drake, Bettina | Colditz, Graham | Wokolorczyk, Dominika | Stephenson, Robert A | Teerlink, Craig | Muller, Heiko | Rothenbacher, Dietrich | Sellers, Thomas A | Lin, Hui-Yi | Slavov, Chavdar | Mitev, Vanio | Lose, Felicity | Srinivasan, Srilakshmi | Maia, Sofia | Paulo, Paula | Lange, Ethan | Cooney, Kathleen A | Antoniou, Antonis | Vincent, Daniel | Bacot, François | Tessier,  | Kote-Jarai, Zsofia | Easton, Douglas F
Nature genetics  2013;45(4):10.1038/ng.2560.
Prostate cancer is the most frequently diagnosed cancer in males in developed countries. To identify common prostate cancer susceptibility alleles, we genotyped 211,155 SNPs on a custom Illumina array (iCOGS) in blood DNA from 25,074 prostate cancer cases and 24,272 controls from the international PRACTICAL Consortium. Twenty-three new prostate cancer susceptibility loci were identified at genome-wide significance (P < 5 × 10−8). More than 70 prostate cancer susceptibility loci, explaining ~30% of the familial risk for this disease, have now been identified. On the basis of combined risks conferred by the new and previously known risk loci, the top 1% of the risk distribution has a 4.7-fold higher risk than the average of the population being profiled. These results will facilitate population risk stratification for clinical studies.
PMCID: PMC3832790  PMID: 23535732
6.  Abnormalities of Calcium Handling Proteins in Skeletal Muscle Mirror those of the Heart in Humans with Heart Failure: a Shared Mechanism? 
Journal of cardiac failure  2012;18(9):724-733.
In the failing human heart, abnormalities of Ca2+ cycling have been described, but there is scant knowledge about Ca2+ handling in the skeletal muscle of humans with HF. We tested the hypothesis that in humans with HF, Ca2+ cycling proteins in skeletal muscle are abnormal.
Methods and Results
Ten advanced HF patients (50.4±3.7 years), and 9 age matched controls underwent vastus lateralis biopsy. Western blot analysis showed that sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a, which is responsible for Ca2+ sequestration into the sarcoplasmic reticulum(SR), was lower in HF vs controls (4.8±0.5vs7.5±0.8AU, p=0.01). Although phospholamban (PLN), which inhibits SERCA2a, was not different in HF vs controls, phosphorylation (SER16 site) of PLN, which relieves this inhibition, was reduced (0.8±0.1vs3.9±0.9AU, p=0.004). Dihydropyridine receptors were reduced in HF, (2.1±0.4vs3.6±0.5AU, p=0.04). We tested the hypothesis that these abnormalities of Ca2+ handling protein content and regulation were due to increased oxidative stress, but oxygen radical scavenger proteins were not elevated in the skeletal muscle of HF patients.
In chronic HF, marked abnormalities of Ca2+ handling proteins are present in skeletal muscle, which mirror those in failing heart tissue. This suggests a common mechanism, such as chronic augmentation of sympathetic activity and autophosphorylation of Ca2+-calmodulin-dependent-protein kinase II.
PMCID: PMC3437990  PMID: 22939042
exercise intolerance; sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a; sympathetic nerve activity; oxidative stress
7.  The Obesity Paradox in Men versus Women with Systolic Heart Failure 
Obesity is common in heart failure (HF) and associated with improved outcomes, often termed the “obesity paradox”. Although fat distribution varies by sex, the role of obesity in the outcomes of women compared to men with HF has not been well-studied. In a cohort of advanced systolic HF patients followed at a single university center, 2718 patients had body mass index (BMI) measured at baseline and 469 HF patients had waist circumference (WC) measured at baseline. Elevated BMI was defined as ≥25 kg/m2. High WC was defined as ≥88 cm in women and ≥102 cm in men. The primary outcome was death, urgent heart transplant, or ventricular assist device placement. Mean age was 53.0 ± 12.4, 25% of subjects were women, and LVEF was 22.9 ± 7.19. In men, 2-year event-free survival was better for high vs. normal BMI (63.2 vs. 53.5% p<0.001) and for high vs. normal WC (78.8% vs. 63.1%, p=0.01). In women, 2-year event-free survival was better in elevated vs. normal BMI groups (67.1% vs. 56.6%, p=0.01), but similar in WC groups. In multivariate analyses, normal BMI and normal WC were associated with higher risk of primary outcome in both men (BMI 1.34, WC 2.02) and women (BMI 1.38, WC 2.99). In advanced HF, high BMI and WC were associated with improved outcomes in both sexes. Further investigation of the interaction between body composition and sex in HF outcomes is warranted.
PMCID: PMC3377856  PMID: 22497678
Cardiomyopathy; mortality; adiposity; gender
8.  Relation between Hemoglobin A1c and Outcomes in Heart Failure Patients with and without Diabetes Mellitus 
The American Journal of Cardiology  2012;109(12):1767-1773.
Among patients with diabetes mellitus (DM) in the general population, elevated glycosylated hemoglobin (HbA1c) increases the risk of developing heart failure (HF). However, in patients with established HF, the association of HbA1c level with outcomes is not well established. This study investigated the relation between HbA1c and outcomes in HF patients with and without diabetes. We studied 845 advanced HF patients followed at the Ahmanson-UCLA Cardiomyopathy Center, stratified by presence (n=358) or absence (n=487) of DM and by DM-specific HbA1c quartiles (Q) (Diabetics: Q1≤6.4%, Q2 6.5–7.2%, Q3 7.3–8.5%, Q4≥8.6%; Non-diabetics: Q1≤5.6%, Q2 5.7–6.0%, Q3 6.1–6.5%, Q4≥6.6%). The primary outcomes analyzed were death and death/urgent heart transplantation (Utx). In the cohort with DM, 2-year event-free survival was 61% and 65% for Q3 and Q4 compared to 48% and 42% in Q1 and Q2 (p=0.005). In the cohort without DM, there was no difference in outcomes by HbA1c Q. Risk-adjusted analysis in the diabetic cohort showed significantly increased hazard ratio (HR) of death/Utx in Q1 and Q2 compared to Q4. For every unit HbA1c increase, there was a 15% decreased HR of death/Utx and all-cause mortality (p=0.006 and p=0.036, respectively). In the cohort without DM, multivariable models revealed similar HR among HbA1c Q. In this cohort of patients with advanced HF, higher HbA1c levels were associated with improved outcomes in patients with diabetes. This relationship was not observed in patients without DM. Further investigations into mechanisms underlying the relationship between HbA1c, DM, and survival in advanced HF are warranted.
PMCID: PMC3367080  PMID: 22459300
diabetes; heart failure; glycosylated hemoglobin; outcomes
9.  RNA-Seq Profiling of Spinal Cord Motor Neurons from a Presymptomatic SOD1 ALS Mouse 
PLoS ONE  2013;8(1):e53575.
Mechanisms involved with degeneration of motor neurons in amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease) are poorly understood, but genetically inherited forms, comprising ∼10% of the cases, are potentially informative. Recent observations that several inherited forms of ALS involve the RNA binding proteins TDP43 and FUS raise the question as to whether RNA metabolism is generally disturbed in ALS. Here we conduct whole transcriptome profiling of motor neurons from a mouse strain, transgenic for a mutant human SOD1 (G85R SOD1-YFP), that develops symptoms of ALS and paralyzes at 5–6 months of age. Motor neuron cell bodies were laser microdissected from spinal cords at 3 months of age, a time when animals were presymptomatic but showed aggregation of the mutant protein in many lower motor neuron cell bodies and manifested extensive neuromuscular junction morphologic disturbance in their lower extremities. We observed only a small number of transcripts with altered expression levels or splicing in the G85R transgenic compared to age-matched animals of a wild-type SOD1 transgenic strain. Our results indicate that a major disturbance of polyadenylated RNA metabolism does not occur in motor neurons of mutant SOD1 mice, suggesting that the toxicity of the mutant protein lies at the level of translational or post-translational effects.
PMCID: PMC3536741  PMID: 23301088
10.  Relation Between Body Mass Index, Exercise Training, and Outcomes in Chronic Systolic Heart Failure 
The American journal of cardiology  2011;108(12):1754-1759.
Exercise training (ET) in heart failure (HF), as demonstrated in the HF-ACTION trial, was associated with improved exercise tolerance and health status, and a trend towards reduced mortality or hospitalizations. This analysis of the HF-ACTION cohort examines the effect of ET in overweight and obese compared to normal HF subjects. 2,314 of 2,331 systolic HF subjects randomized to aerobic ET vs. usual care in HF-ACTION were analyzed to determine the effect of ET on all cause mortality, hospitalizations, exercise parameters, quality of life (QOL), and body weight changes by subgroups of body mass index (BMI). Strata included normal weight (BMI 18.5 – 24.9 kg/m2), overweight (BMI 25.0 – 29.9 kg/m2), obese I (BMI 30 – 34.9 kg/m2), obese II (BMI 35-39.9 kg/m2), and obese III (BMI ≥ 40 kg/m2). At enrollment, 19.4% of subjects were normal weight, 31.3% overweight, and 49.4% obese. Higher BMI was associated with a non-significant increase in all cause mortality or hospitalization. ET was associated with non-significant reductions in all cause mortality or hospitalization in each weight category (HR 0.98, 0.95, 0.92, 0.89, and 0.86 in normal weight, overweight, obese I, obese II, and obese III categories, respectively [all p>0.05]). Modeled improvement in exercise capacity (peak oxygen consumption) and QOL in the ET group was seen in all BMI categories. In conclusion, aerobic ET in HF was associated with a non-significant trend towards decreased mortality and hospitalizations and a significant improvement in QOL across the range of BMI categories.
PMCID: PMC3229667  PMID: 21907317
heart failure; body mass index; exercise
11.  Maintaining success, reducing treatment burden, focusing on survivorship: highlights from the third European consensus conference on diagnosis and treatment of germ-cell cancer 
Annals of Oncology  2012;24(4):878-888.
In November 2011, the Third European Consensus Conference on Diagnosis and Treatment of Germ-Cell Cancer (GCC) was held in Berlin, Germany. This third conference followed similar meetings in 2003 (Essen, Germany) and 2006 (Amsterdam, The Netherlands) [Schmoll H-J, Souchon R, Krege S et al. European consensus on diagnosis and treatment of germ-cell cancer: a report of the European Germ-Cell Cancer Consensus Group (EGCCCG). Ann Oncol 2004; 15: 1377–1399; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part I. Eur Urol 2008; 53: 478–496; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part II. Eur Urol 2008; 53: 497–513]. A panel of 56 of 60 invited GCC experts from all across Europe discussed all aspects on diagnosis and treatment of GCC, with a particular focus on acute and late toxic effects as well as on survivorship issues.
The panel consisted of oncologists, urologic surgeons, radiooncologists, pathologists and basic scientists, who are all actively involved in care of GCC patients. Panelists were chosen based on the publication activity in recent years. Before the meeting, panelists were asked to review the literature published since 2006 in 20 major areas concerning all aspects of diagnosis, treatment and follow-up of GCC patients, and to prepare an updated version of the previous recommendations to be discussed at the conference. In addition, ∼50 E-vote questions were drafted and presented at the conference to address the most controversial areas for a poll of expert opinions. Here, we present the main recommendations and controversies of this meeting. The votes of the panelists are added as online supplements.
PMCID: PMC3603440  PMID: 23152360
consensus conference; diagnosis; germ-cell cancer; late toxic effects; long-term follow-up; treatment
12.  Statins Do Not Significantly Affect Muscle Sympathetic Nerve Activity in Humans with Non-Ischemic Heart Failure: A Double-Blind Placebo Controlled Trial 
Journal of cardiac failure  2011;17(11):879-886.
HMG-CoA reductase inhibitors (statins) have been shown to reduce sympathetic nervous system (SNS) activation in experimental heart failure (HF). However, this potential mechanism of action of statins in HF has not been well studied in humans.
Methods and Results
Twenty-six patients with non-ischemic, systolic HF (left ventricular ejection fraction [LVEF] ≤ 35%) were randomized to atorvastatin (10mg) vs. placebo for 3 months. Pre- and post- treatment testing included echocardiography, laboratories, quality of life (QOL) questionnaires, and peroneal nerve muscle sympathetic nerve activity (MSNA) via microneurography. Eighteen subjects had technically adequate pre- and post- MSNA tracings. The cohort was 65% male, 81% NYHA II, LVEF 26 ± 6%, and LDL cholesterol 108 ± 26 mg/dl. Baseline MSNA was 41 ± 2 bursts / minute. LDL significantly decreased in the atorvastatin (−36.8%) vs. placebo (−0.1%) group (p<0.0001). However, there was no significant change in MSNA (−16.2% vs. – 2.5%), LVEF, B-type natriuretic peptide, or QOL score in the atorvastatin compared to placebo group.
Short-term statin therapy in patients with non-ischemic HF does not result in a significant decrease in SNS activation as measured by MSNA. These findings are consistent with the neutral outcomes of large clinical trials of statins in HF.
PMCID: PMC3206298  PMID: 22041323
heart failure; lipids; nervous system; sympathetic
13.  Translational diffusion of macromolecular assemblies measured using transverse relaxation-optimized PFG-NMR 
Journal of the American Chemical Society  2011;133(41):16354-16357.
In structural biology, pulsed field gradient (PFG) NMR for characterization of size and hydrodynamic parameters of macromolecular solutes has the advantage over other techniques that the measurements can be recorded with identical solution conditions as used for NMR structure determination or for crystallization trials. This paper describes two transverse relaxation-optimized (TRO) 15N-filtered PFG stimulated-echo (STE) experiments for studies of macromolecular translational diffusion in solution, 1H-TRO-STE and 15N-TRO-STE, which include CRINEPT and TROSY elements. Measurements with mixed micelles of the Escherichia coli outer membrane protein X (OmpX) and the detergent Fos-10 were used for a systematic comparison of 1H-TRO-STE and 15N-TRO-STE with conventional 15N-filtered STE experimental schemes. The results provide an extended platform for evaluating the NMR experiments available for diffusion measurements in structural biology projects with molecular particles of different size ranges. An initial application of the 15N-TRO-STE experiment with very long diffusion delays showed that the tedradecamer structure of the 800 kDa Thermus thermophilus chaperonin GroEL is preserved in aqueous solution over the temperature range 25–60°C.
PMCID: PMC3358227  PMID: 21919531
14.  Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid 
The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining.
PMCID: PMC3177974  PMID: 21890355
GroEL; chaperonin; chaperone; protein localization; unnatural amino acid; tRNA
15.  Seven novel prostate cancer susceptibility loci identified by a multi-stage genome-wide association study 
Kote-Jarai, Zsofia | Olama, Ali Amin Al | Giles, Graham G. | Severi, Gianluca | Schleutker, Johanna | Weischer, Maren | Canzian, Frederico | Riboli, Elio | Key, Tim | Gronberg, Henrik | Hunter, David J. | Kraft, Peter | Thun, Michael J | Ingles, Sue | Chanock, Stephen | Albanes, Demetrius | Hayes, Richard B | Neal, David E. | Hamdy, Freddie C. | Donovan, Jenny L. | Pharoah, Paul | Schumacher, Fredrick | Henderson, Brian E. | Stanford, Janet L. | Ostrander, Elaine A. | Sorensen, Karina Dalsgaard | Dörk, Thilo | Andriole, Gerald | Dickinson, Joanne L. | Cybulski, Cezary | Lubinski, Jan | Spurdle, Amanda | Clements, Judith A. | Chambers, Suzanne | Aitken, Joanne | Frank Gardiner, R. A. | Thibodeau, Stephen N. | Schaid, Dan | John, Esther M. | Maier, Christiane | Vogel, Walther | Cooney, Kathleen A. | Park, Jong Y. | Cannon-Albright, Lisa | Brenner, Hermann | Habuchi, Tomonori | Zhang, Hong-Wei | Lu, Yong-Jie | Kaneva, Radka | Muir, Ken | Benlloch, Sara | Leongamornlert, Daniel A. | Saunders, Edward J. | Tymrakiewicz, Malgorzata | Mahmud, Nadiya | Guy, Michelle | O’Brien, Lynne T. | Wilkinson, Rosemary A. | Hall, Amanda L. | Sawyer, Emma J. | Dadaev, Tokhir | Morrison, Jonathan | Dearnaley, David P. | Horwich, Alan | Huddart, Robert A. | Khoo, Vincent S. | Parker, Christopher C. | Van As, Nicholas | Woodhouse, Christopher J. | Thompson, Alan | Christmas, Tim | Ogden, Chris | Cooper, Colin S. | Lophatonanon, Aritaya | Southey, Melissa C. | Hopper, John L. | English, Dallas | Wahlfors, Tiina | Tammela, Teuvo LJ | Klarskov, Peter | Nordestgaard, Børge G. | Røder, M. Andreas | Tybjærg-Hansen, Anne | Bojesen, Stig E. | Travis, Ruth | Campa, Daniele | Kaaks, Rudolf | Wiklund, Fredrik | Aly, Markus | Lindstrom, Sara | Diver, W Ryan | Gapstur, Susan | Stern, Mariana C | Corral, Roman | Virtamo, Jarmo | Cox, Angela | Haiman, Christopher A. | Le Marchand, Loic | FitzGerald, Liesel | Kolb, Suzanne | Kwon, Erika M. | Karyadi, Danielle M. | Orntoft, Torben Falck | Borre, Michael | Meyer, Andreas | Serth, Jürgen | Yeager, Meredith | Berndt, Sonja I. | Marthick, James R | Patterson, Briony | Wokolorczyk, Dominika | Batra, Jyotsna | Lose, Felicity | McDonnell, Shannon K | Joshi, Amit D. | Shahabi, Ahva | Rinckleb, Antje E. | Ray, Ana | Sellers, Thomas A. | Lin, Huo-Yi | Stephenson, Robert A | Farnham, James | Muller, Heiko | Rothenbacher, Dietrich | Tsuchiya, Norihiko | Narita, Shintaro | Cao, Guang-Wen | Slavov, Chavdar | Mitev, Vanio | Easton, Douglas F. | Eeles, Rosalind A.
Nature Genetics  2011;43(8):785-791.
Prostate cancer (PrCa) is the most frequently diagnosed male cancer in developed countries. To identify common PrCa susceptibility alleles, we conducted a multi-stage genome-wide association study and previously reported the results of the first two stages, which identified 16 novel susceptibility loci for PrCa. Here we report the results of stage 3 in which we evaluated 1,536 SNPs in 4,574 cases and 4,164 controls. Ten novel association signals were followed up through genotyping in 51,311 samples in 30 studies through the international PRACTICAL consortium. In addition to previously reported loci, we identified a further seven new prostate cancer susceptibility loci on chromosomes 2p, 3q, 5p, 6p, 12q and Xq (P=4.0 ×10−8 to P=2.7 ×10−24). We also identified a SNP in TERT more strongly associated with PrCa than that previously reported. More than 40 PrCa susceptibility loci, explaining ~25% of the familial risk in this disease, have now been identified.
PMCID: PMC3396006  PMID: 21743467
16.  ATP-Triggered Conformational Changes Delineate Substrate-Binding and -Folding Mechanics of the GroEL Chaperonin 
Cell  2012;149(1):113-123.
The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the “power stroke” that ejects substrate into the folding chamber.
Graphical Abstract
► Single-particle cryo-EM identifies seven ATP-bound structures of GroEL chaperonin ► Analysis of structures suggests trajectory of domain movements triggered by ATP binding ► The structures delineate a mechanism for mechanical unfolding of polypeptide substrate ► Model suggests an intermediate conformation binding substrate and GroES simultaneously
Cryo-EM analysis reveals a series of conformational steps through which GroEL binds polypeptide substrates, stretches and unfolds them, and then moves them into a folding chamber that promotes adoption of the native state.
PMCID: PMC3326522  PMID: 22445172
17.  Epidemiology and risk profile of heart failure 
Nature reviews. Cardiology  2010;8(1):30-41.
Heart failure (HF) is a major public health issue, with a prevalence of over 5.8 million in the USA, and over 23 million worldwide, and rising. The lifetime risk of developing HF is one in five. Although promising evidence shows that the age-adjusted incidence of HF may have plateaued, HF still carries substantial morbidity and mortality, with 5-year mortality that rival those of many cancers. HF represents a considerable burden to the health-care system, responsible for costs of more than $39 billion annually in the USA alone, and high rates of hospitalizations, readmissions, and outpatient visits. HF is not a single entity, but a clinical syndrome that may have different characteristics depending on age, sex, race or ethnicity, left ventricular ejection fraction (LVEF) status, and HF etiology. Furthermore, pathophysiological differences are observed among patients diagnosed with HF and reduced LVEF compared with HF and preserved LVEF, which are beginning to be better appreciated in epidemiological studies. A number of risk factors, such as ischemic heart disease, hypertension, smoking, obesity, and diabetes, among others, have been identified that both predict the incidence of HF as well as its severity. In this Review, we discuss key features of the epidemiology and risk profile of HF.
PMCID: PMC3033496  PMID: 21060326
18.  Double mutant MBP refolds at same rate in free solution as inside the GroEL/GroES chaperonin chamber when aggregation in free solution is prevented 
FEBS letters  2011;585(12):1969-1972.
Under “permissive” conditions at 25°C, the chaperonin substrate protein DM-MBP refolds 5–10 times more rapidly in the GroEL/GroES folding chamber than in free solution. This has been suggested to indicate that the chaperonin accelerates polypeptide folding by entropic effects of close confinement. Here, using native-purified DM-MBP, we show that the different rates of refolding are due to reversible aggregation of DM-MBP while folding free in solution, slowing its kinetics of renaturation: the protein exhibited concentration-dependent refolding in solution, with aggregation directly observed by dynamic light scattering. When refolded in chloride-free buffer, however, dynamic light scattering was eliminated, refolding became concentration-independent, and the rate of refolding became the same as that in GroEL/GroES. The GroEL/GroES chamber thus appears to function passively toward DM-MBP.
PMCID: PMC3144026  PMID: 21609718
DM-MBP; MBP; GroEL; GroES; aggregation
19.  A Chaperonin Subunit with Unique Structures Is Essential for Folding of a Specific Substrate 
PLoS Biology  2011;9(4):e1001040.
Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60β4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60β1–β3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60β subunits cannot complement the function of Cpn60β4. Furthermore, the unique C-terminus of Cpn60β4 is required for the full activity of the unique Cpn60 complex containing Cpn60β4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates.
Author Summary
Chaperonins assist the folding of some nascent and denatured proteins to their native, functional forms. Each chaperonin consists of a pair of protein complexes resembling two stacked toroids; folding occurs inside the toroid cavity. Chaperonins are ubiquitous in both bacteria and more complex nucleated cells, as well as in the intracellular organelles that have evolved from bacteria by endosymbiosis: mitochondria and, in plants, chloroplasts. They are indispensable for cellular function. Many different chaperonin subunits have evolved in various species of bacteria as well as in most mitochondria and chloroplasts. The physiological and functional relevance of these multiple chaperonin subunits is poorly understood, however. In this study, we have characterized the minor chaperonin subunit Cpn60β4 from Arabidopsis chloroplasts, which differs in structure from other chloroplast chaperonins. When the Cpn60β4 gene is defective, the plants fail to accumulate one protein complex in particular: the chloroplast NADH dehydrogenase-like complex (NDH). We discovered that Cpn60β4 forms a complex with other Cpn60 α and β subunits and that this complex is essential for the folding of the NDH subunit NdhH. Cpn60β4 has a unique protein “tail” that is required for the efficient folding of NdhH. Our findings suggest that Cpn60β4 has evolved with distinctive structural features that facilitate the folding of one specific substrate and that this strategy is used by plants to satisfy their conflicting requirements for chaperonins with both specialized and general functions.
PMCID: PMC3071376  PMID: 21483722
20.  Glucose, Obesity, Metabolic Syndrome, and Diabetes: Relevance to Incidence of Heart Failure 
Heart failure (HF) is common, results in poor clinical outcomes, and is associated with large health-care costs. The incidence of HF continues to rise, with approximately 670,000 new cases per year and a 20% lifetime risk of HF for persons 40 years and older in the United States. Risk factors for HF have been identified and thus preventative strategies should have a positive effect on disease burden, morbidity, and mortality. Although coronary artery disease and hypertension have traditionally been considered among the most important modifiable risk factors for the development of HF, recent studies have highlighted the importance of increasingly prevalent metabolic risk factors – glucose, diabetes, obesity, and the metabolic syndrome. This paper will present evidence for the link between glucose, diabetes, obesity, metabolic syndrome and incident HF. Furthermore, we will discuss how risk factor modification and other preventive therapies may help curb the rising incidence of HF.
PMCID: PMC2834416  PMID: 20117431
Glucose; Obesity; Heart Failure; Metabolic Syndrome; Diabetes
21.  Target RNA-directed trimming and tailing of small silencing RNAs 
Science (New York, N.Y.)  2010;328(5985):1534-1539.
In Drosophila, microRNAs (miRNAs) typically guide Argonaute1 to repress mRNA, whereas small interfering RNAs (siRNAs) guide Argonaute2 to destroy viral and transposon RNA. Unlike siRNAs, miRNAs rarely base pair extensively to the mRNAs they regulate. We find that extensive complementarity between a target RNA and an Argonaute1-bound miRNA triggers miRNA tailing and 3′-to-5′ trimming. In flies, Argonaute2-bound small RNAs—but not those bound to Argonaute1—bear a 2′-O-methyl group at their 3′ ends. This modification blocks target-directed small RNA remodeling: in flies lacking Hen1, the enzyme that adds the 2′-O-methyl group, Argonaute2-associated siRNAs are tailed and trimmed. Target-complementarity also affects small RNA stability in human cells. These results provide an explanation for the partial complementarity between animal miRNAs and their targets.
PMCID: PMC2902985  PMID: 20558712
22.  Endogenous siRNAs Derived from Transposons and mRNAs in Drosophila Somatic Cells 
Science (New York, N.Y.)  2008;320(5879):1077-1081.
Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line.
PMCID: PMC2953241  PMID: 18403677
23.  The Relationship Between Body Mass Index and Cardiopulmonary Exercise Testing in Chronic Systolic Heart Failure 
American heart journal  2009;158(4 Suppl):S31-S36.
Cardiopulmonary exercise testing (CPX) in patients with systolic heart failure (HF) is important for determining HF prognosis and helping guide timing of heart transplantation. Although approximately 20–30% of patients with HF are obese (body mass index [BMI]>30kg/m2), the impact of BMI on CPX results is not well established. The objective of the present study was to assess the relationship between BMI and CPX variables, including peak oxygen uptake, VO2 at ventilatory threshold, O2 pulse, and ventilation / carbon dioxide production ratio.
Consecutive systolic HF patients (n=2324) enrolled in the Heart Failure and A Controlled Trial Investigating Outcomes of Exercise Training (HF-ACTION) trial who had baseline BMI recorded were included in the present study. Subjects were divided into strata based on BMI: underweight (BMI< 18.5 kg/m2), normal weight (BMI 18.5 – 24.9 kg/m2), overweight (BMI 25.0 – 29.9 kg/m2), obese I (BMI 30 – 34.9 kg/m2), obese II (BMI 35–39.9 kg/m2), and obese III (BMI ≥ 40 kg/m2).
Obese III, but not overweight, obese I, or obese II, was associated with decreased peak oxygen uptake (mL/kg/min) compared to normal weight status. Increasing BMI category was inversely related to ventilation / carbon dioxide production (VE/VCO2) ratio (p< 0.0001). On multivariable analysis, BMI was a significant independent predictor of peak oxygen uptake (partial R2 = 0.07, p< 0.0001) and VE/VCO2 slope (partial R2 = 0.03, p< 0.0001) in patients with chronic systolic HF.
BMI is significantly associated with key CPX fitness variables in HF patients. The influence of BMI on the prognostic value of CPX in HF requires further evaluation in longitudinal studies.
PMCID: PMC2762950  PMID: 19782786
24.  Identification of seven new prostate cancer susceptibility loci through a genome-wide association study 
Eeles, Rosalind A. | Kote-Jarai, Zsofia | Olama, Ali Amin Al | Giles, Graham G. | Guy, Michelle | Severi, Gianluca | Muir, Kenneth | Hopper, John L. | Henderson, Brian E. | Haiman, Christopher A. | Schleutker, Johanna | Hamdy, Freddie C. | Neal, David E. | Donovan, Jenny L. | Stanford, Janet L. | Ostrander, Elaine A. | Ingles, Sue A. | John, Esther M. | Thibodeau, Stephen N. | Schaid, Daniel | Park, Jong Y. | Spurdle, Amanda | Clements, Judith | Dickinson, Joanne L. | Maier, Christiane | Vogel, Walther | Dörk, Thilo | Rebbeck, Timothy R. | Cooney, Kathleen A. | Cannon-Albright, Lisa | Chappuis, Pierre O. | Hutter, Pierre | Zeegers, Maurice | Kaneva, Radka | Zhang, Hong-Wei | Lu, Yong-Jie | Foulkes, William D. | English, Dallas R. | Leongamornlert, Daniel A. | Tymrakiewicz, Malgorzata | Morrison, Jonathan | Ardern-Jones, Audrey T. | Hall, Amanda L. | O’Brien, Lynne T. | Wilkinson, Rosemary A. | Saunders, Edward J. | Page, Elizabeth C. | Sawyer, Emma J. | Edwards, Stephen M. | Dearnaley, David P. | Horwich, Alan | Huddart, Robert A. | Khoo, Vincent S. | Parker, Christopher C. | Van As, Nicholas | Woodhouse, Christopher J. | Thompson, Alan | Christmas, Tim | Ogden, Chris | Cooper, Colin S. | Southey, Melissa C. | Lophatananon, Artitaya | Liu, Jo-Fen | Kolonel, Laurence N. | Le Marchand, Loic | Wahlfors, Tiina | Tammela, Teuvo L. | Auvinen, Anssi | Lewis, Sarah J. | Cox, Angela | FitzGerald, Liesel M. | Koopmeiners, Joseph S. | Karyadi, Danielle M. | Kwon, Erika M. | Stern, Mariana C. | Corral, Roman | Joshi, Amit D. | Shahabi, Ahva | McDonnell, Shannon K. | Sellers, Thomas A | Pow-Sang, Julio | Chambers, Suzanne | Aitken, Joanne | Gardiner, R.A. (Frank) | Batra, Jyotsna | Kedda, Mary Anne | Lose, Felicity | Polanowski, Andrea | Patterson, Briony | Serth, Jürgen | Meyer, Andreas | Luedeke, Manuel | Stefflova, Klara | Ray, Anna M. | Lange, Ethan M. | Farnham, Jim | Khan, Humera | Slavov, Chavdar | Mitkova, Atanaska | Cao, Guangwen | Easton, Douglas F.
Nature genetics  2009;41(10):1116-1121.
Prostate cancer (PrCa) is the most frequently diagnosed male cancer in developed countries. To identify common PrCa susceptibility alleles, we have previously conducted a genome-wide association study in which 541, 129 SNPs were genotyped in 1,854 PrCa cases with clinically detected disease and 1,894 controls. We have now evaluated promising associations in a second stage, in which we genotyped 43,671 SNPs in 3,650 PrCa cases and 3,940 controls, and a third stage, involving an additional 16,229 cases and 14,821 controls from 21 studies. In addition to previously identified loci, we identified a further seven new prostate cancer susceptibility loci on chromosomes 2, 4, 8, 11, and 22 (P=1.6×10−8 to P=2.7×10−33).
PMCID: PMC2846760  PMID: 19767753
25.  ATP-triggered ADP release from the asymmetric chaperonin GroEL/GroES/ADP7 is not the rate-limiting step of the GroEL/GroES reaction cycle 
FEBS letters  2010;584(5):951-953.
The GroEL/GroES protein folding chamber is formed and dissociated by ATP binding and hydrolysis. ATP hydrolysis in the GroES-bound (cis) ring gates entry of ATP into the opposite unoccupied trans ring, which allosterically ejects cis ligands. While earlier studies suggested that hydrolysis of cis ATP is the rate limiting step of the cycle (t½ ~10s), a recent study suggested that ADP release from the cis ring may be rate-limiting (t½ ~15–20 sec). Here we have measured ADP release using a coupled enzyme assay and observed a t½ for release of ≤4–5 seconds, indicating that this is not the rate-limiting step of the reaction cycle.
PMCID: PMC2849271  PMID: 20083109
GroEL; GroES; ATP; ADP; Nucleotide cycle; Coupled enzyme assay

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