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1.  The structure and function of G-protein-coupled receptors 
Nature  2009;459(7245):356-363.
G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants, and so have great potential as therapeutic targets for a broad spectrum of diseases. They are also fascinating molecules from the perspective of membrane-protein structure and biology. Great progress has been made over the past three decades in understanding diverse GPCRs, from pharmacology to functional characterization in vivo. Recent high-resolution structural studies have provided insights into the molecular mechanisms of GPCR activation and constitutive activity.
PMCID: PMC3967846  PMID: 19458711
2.  Hepatocyte Growth Factor and the Risk of Developing Ischemic Stroke Among Postmenopausal Women: Results from the Women’s Health Initiative 
Hepatocyte growth factor (HGF) is a potent angiogenic factor and may play a role in the development and progression of atherosclerotic lesions, the underlying mechanism of cardiovascular disease. However, there have been no prospective studies examining the relationship between HGF levels and risk of stroke.
Methods and Results
We conducted a nested case-control study (972 incident stroke cases and 1:1 age- and race-matched controls) to prospectively evaluate the association between plasma HGF and risk of ischemic stroke within the Women’s Health Initiative Observational Study, a cohort of postmenopausal women aged 50–79 years. Baseline HGF levels were correlated positively with body mass index (BMI), systolic blood pressure, low-density lipoprotein cholesterol, insulin resistance, and inflammatory markers such as C-reactive protein, and inversely with high-density lipoprotein cholesterol (all P-values <0.05). Baseline HGF levels were higher among cases than controls (geometric means 601.8 vs. 523.2 pg/mL, p = 0.003). Furthermore, the risk of incident ischemic stroke was significantly greater amongst women in the highest versus lowest quartile of plasma HGF levels (odds ratio [OR] = 1.46; 95% confidence interval [CI]: 1.12–1.91; Ptrend = 0.003), in a conditional logistic regression model that adjusted for BMI. These results were only slightly attenuated after further adjustment for additional stroke risk factors (OR=1.39; 95% CI=1.04–1.85, Ptrend=0.023).
Circulating levels of HGF are associated with an increased risk of incident ischemic stroke, independent of obesity and other risk factors for cardiovascular disease among postmenopausal women aged 50–79 years.
PMCID: PMC3903044  PMID: 20203323
Hepatocyte growth factor; ischemic stroke; women
3.  Lipid and Lipoprotein Biomarkers and the Risk of Ischemic Stroke in Postmenopausal Women 
Few studies simultaneously investigated lipids and lipoprotein biomarkers as predictors of ischemic stroke. The value of these biomarkers as independent predictors of ischemic stroke remains controversial.
We conducted a prospective nested case-control study among postmenopausal women from the Women’s Health Initiative Observational Study to assess the relationship between fasting lipids (total cholesterol, LDL-C, HDL-C, and triglycerides), lipoproteins (LDL, HDL and VLDL particle number and size, IDL particle number, and lipoprotein [a]) and risk of ischemic stroke. Among women free of stroke at baseline, 774 ischemic stroke patients were matched according to age and race to controls using a 1:1 ratio.
In bivariate analysis, baseline triglycerides (P<0.001), IDL particles (P<0.01), LDL particles (P<0.01), VLDL triglyceride (P<0.001), VLDL particles (P<0.01), VLDL size (P<0.001), LDL size (P=0.03), and total/HDL cholesterol ratio (P<0.01) were significantly higher among women with incident ischemic stroke, while levels of HDL-C (P<0.01) and HDL size (P<0.01) were lower. No significant baseline difference for total cholesterol (P=0.15), LDL-C (P=0.47), and lipoprotein (a) (P=0.11) was observed. In multivariable analysis, triglycerides, (OR for the highest vs lowest quartile, 1.56; 95% CI, 1.13-2.17, P for trend =0.02), VLDL size (OR 1.59, 95% CI, 1.10-2.28, P for trend =0.03) and IDL particle number (OR 1.46, 95% CI, 1.04-2.04, P for trend =0.02) were significantly associated with ischemic stroke.
Among a panel of lipid and lipoprotein biomarkers, baseline triglycerides, VLDL size and IDL particle number were significantly associated with incident ischemic stroke in postmenopausal women.
PMCID: PMC3547588  PMID: 22308251
Lipids; Lipoproteins; Ischemic Stroke; Women; Triglycerides
4.  Long-Term Post-Stroke Changes Include Myelin Loss, Specific Deficits in Sensory and Motor Behaviors and Complex Cognitive Impairment Detected Using Active Place Avoidance 
PLoS ONE  2013;8(3):e57503.
Persistent neurobehavioral deficits and brain changes need validation for brain restoration. Two hours middle cerebral artery occlusion (tMCAO) or sham surgery was performed in male Sprague-Dawley rats. Neurobehavioral and cognitive deficits were measured over 10 weeks included: (1) sensory, motor, beam balance, reflex/abnormal responses, hindlimb placement, forepaw foot fault and cylinder placement tests, and (2) complex active place avoidance learning (APA) and simple passive avoidance retention (PA). Electroretinogram (ERG), hemispheric loss (infarction), hippocampus CA1 neuronal loss and myelin (Luxol Fast Blue) staining in several fiber tracts were also measured. In comparison to Sham surgery, tMCAO surgery produced significant deficits in all behavioral tests except reflex/abnormal responses. Acute, short lived deficits following tMCAO were observed for forelimb foot fault and forelimb cylinder placement. Persistent, sustained deficits for the whole 10 weeks were exhibited for motor (p<0.001), sensory (p<0.001), beam balance performance (p<0.01) and hindlimb placement behavior (p<0.01). tMCAO produced much greater and prolonged cognitive deficits in APA learning (maximum on last trial of 604±83% change, p<0.05) but only a small, comparative effect on PA retention. Hemispheric loss/atrophy was measured 10 weeks after tMCAO and cross-validated by two methods (e.g., almost identical % ischemic hemispheric loss of 33.4±3.5% for H&E and of 34.2±3.5% for TTC staining). No visual dysfunction by ERG and no hippocampus neuronal loss were detected after tMCAO. Fiber tract damage measured by Luxol Fast Blue myelin staining intensity was significant (p<0.01) in the external capsule and striatum but not in corpus callosum and anterior commissure. In summary, persistent neurobehavioral deficits were validated as important endpoints for stroke restorative research in the future. Fiber myelin loss appears to contribute to these long term behavioral dysfunctions and can be important for cognitive behavioral control necessary for complex APA learning.
PMCID: PMC3591420  PMID: 23505432
5.  Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor 
Nature  2012;482(7386):552-556.
Acetylcholine (ACh), the first neurotransmitter to be identified1, exerts many of its physiological actions via activation of a family of G protein-coupled receptors (GPCRs) known as muscarinic ACh receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G protein coupling preference and the physiological responses they mediate.2–4 Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences.5–6 We describe here the structure of the Gq/11-coupled M3 mAChR bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the Gi/o-coupled M2 receptor, offers new possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows the first structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and raise additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer new insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.
PMCID: PMC3529910  PMID: 22358844
6.  A Novel Calpain Inhibitor for Treatment of Transient Retinal Ischemia in the Rat 
Neuroreport  2011;22(13):633-636.
Following an acute ischemia/reperfusion of the rat retina, the activation of cytotoxic proteases, including calpain, results in necrosis and apoptosis of retinal ganglion cells resulting in their degeneration. Using a systemically administered calpain inhibitor that crosses the Blood-Retinal-Barrier would provide for novel systemic intervention that protects the retina from acute injury and lost function. Here we study a novel calpain peptide inhibitor, cysteic–leucyl–argininal (CYLA), in an in vivo rat model of retinal ischemia to determine functional protection using electroretinography. The CYLA-prodrug was administered intraperitoneally before and/or after ischemia-reperfusion at concentrations of 20-40 mg/kg. We found that administering 20 mg/kg CYLA even only after ischemia provides significant preservation of retinal function.
PMCID: PMC3156414  PMID: 21841454
7.  Thrombopoietin protects the brain and improves sensorimotor functions: reduction of stroke-induced MMP-9 upregulation and blood–brain barrier injury 
This study was conducted to determine the protective efficacy and mechanisms of thrombopoietin (TPO) intervention in experimental focal stroke. Male rats underwent 2 hours of left middle cerebral artery occlusion (MCAO) followed by 22 hours of reperfusion. Vehicle or TPO (0.03 to 1.00 μg/kg) was administered intravenously immediately after reperfusion. Brain infarct and swelling, neurologic deficits, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), TPO and c-Mpl (TPO receptor) mRNA, MMP-9 enzyme activity and protein expression, and the integrity of the blood–brain barrier (BBB) were subsequently measured. MCAO reperfusion produced a large infarct and swelling after stroke. Thrombopoietin significantly reduced these in a dose-dependent manner. The most effective TPO dose, 0.1 μg/kg, when administrated immediately or 2 hours after reperfusion, significantly reduced infarct and swelling and ameliorated neurologic deficits after stroke. Stroke-induced increases in cortical MMP-9 mRNA, enzyme activity and protein expression, TIMP-1 mRNA, and Evans blue extravasation were reduced by TPO intervention. Thrombopoietin did not alter cortical TPO or c-Mpl mRNA expression, blood pressure, heart rate, blood hematocrit, or platelets. This is the first demonstration of TPO's efficacy in reducing ischemic brain injury and improving functional outcome, partly by inhibiting the stroke-induced increase in MMP-9 and the early, negative effects on the BBB.
PMCID: PMC3063625  PMID: 20877384
blood–brain barrier; brain infarction; focal ischemia; matrix metalloproteinase-9; neurologic deficits; stroke; thrombopoietin
8.  Structure of a nanobody-stabilized active state of the β2 adrenoceptor 
Nature  2011;469(7329):175-180.
G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviors in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β2 adrenergic receptor (β2AR) that exhibits G protein-like behavior, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β2AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.
PMCID: PMC3058308  PMID: 21228869
9.  Structure and Function of an Irreversible Agonist-β2 Adrenoceptor complex 
Nature  2011;469(7329):236-240.
G protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signaling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs1, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β2 adrenergic receptor (β2AR) as a guide, we designed a β2AR agonist that can be covalently tethered to a specific site on the receptor through a disulfide bond. The covalent β2AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β2AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method2, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper3) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.
PMCID: PMC3074335  PMID: 21228876
10.  Inflammation and hemostasis biomarkers for predicting stroke in postmenopausal women: The Women’s Health Initiative Observational Study 
Inflammatory and hemostasis-related biomarkers may identify women at risk of stroke.
Hormones and Biomarkers Predicting Stroke is a study of ischemic stroke among postmenopausal women participating in the Women’s Health Initiative Observational Study (n = 972 case-control pairs). A Biomarker Risk Score was derived from levels of seven inflammatory and hemostasis-related biomarkers that appeared individually to predict risk of ischemic stroke: C-reactive protein, interleukin-6, tissue plasminogen activator, D-dimer, white blood cell count, neopterin, and homocysteine. The c index was used to evaluate discrimination.
Of all the individual biomarkers examined, C-reactive protein emerged as the only independent single predictor of ischemic stroke (adjusted odds ratio comparing Q4 versus Q1 = 1.64, 95% confidence interval: 1.15–2.32, p = 0.01) after adjustment for other biomarkers and standard stroke risk factors. The Biomarker Risk Score identified a gradient of increasing stroke risk with a greater number of elevated inflammatory/hemostasis biomarkers, and improved the c index significantly compared with standard stroke risk factors (p = 0.02). Among the subset of individuals who met current criteria for “high risk” levels of C-reactive protein (> 3.0 mg/L), the Biomarker Risk Score defined an approximately two-fold gradient of risk. We found no evidence for a relationship between stroke and levels of E-selectin, fibrinogen, tumor necrosis factor-alpha, vascular cell adhesion molecule-1, prothrombin fragment 1+2, Factor VIIC, or plasminogen activator inhibitor-1 antigen (p >0.15).
The findings support the further exploration of multiple-biomarker panels to develop approaches for stratifying an individual’s risk of stroke.
PMCID: PMC3077422  PMID: 18984425
stroke; epidemiology; women
11.  Involvement of Erythropoietin in Retinal Ischemic Preconditioning 
Anesthesiology  2009;110(4):774-780.
The purpose of this study was to examine the role of erythropoietin in retinal ischemic preconditioning (IPC).
Rats were subjected to retinal ischemia after IPC. Electroretinography assessed functional recovery after ischemia; retinal sections were examined to determine loss of retinal ganglion cells, and Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling was used to assess apoptosis. Levels of downstream mediators were measured in retinal homogenates by Western blotting. To assess the involvement of erythropoietin in IPC, we measured levels of erythropoietin and its receptor (EPO-R) in retinal homogenates following IPC, using Western blotting. To examine erythropoietin’s role in IPC, we studied the impact of blocking erythropoietin via intravitreal injection of soluble EPO-R (sEPO-R) before IPC.
Erythropoietin levels did not change following IPC, but EPO-R increased. Intravitreal injection of sEPO-R significantly attenuated both the functional and histological neuroprotection produced by IPC in comparison to control injection of denatured sEPO-R. Apoptotic damage after ischemia was enhanced in the sEPO-R treated retinas as indicated by fluorescent Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling. Phosphorylated extracellular-signal-regulated kinase (ERK) and heat shock protein 27 (Hsp27), but not protein kinase B (Akt), upregulated in denatured sEPO-R treated retinae, were attenuated in eyes injected with sEPO-R.
These results indicate that EPO-R upregulation is a critical component of the functional, histological, and anti-apoptotic protective effect of ischemic preconditioning on ischemia in the retina and that several downstream effectors may be involved in the neuroprotective actions of erythropoietin.
PMCID: PMC2891304  PMID: 19322943
12.  Harmful effect of kainic acid on brain ischemic damage is not related to duration of status epilepticus 
Status epilepticus is common in infants and may have long-term consequences on the brain persisting into adulthood. Vascular ischemia is a common cause of stroke in adulthood. The extent of stroke in 15-day-old rats is larger when previously exposed to kainic acid-induced status epilepticus. In this paper, we assess whether shortening the duration of seizures modifies subsequent susceptibility to middle cerebral artery occlusion. We administered pentobarbital 50 mg/kg to abort seizures after 1 h. Although administration of pentobarbital aborted seizures, it had no effect on volume of infarction following ischemia. This study indicates that there is dissociation between stopping status epilepticus and modifying its long-term consequences.
PMCID: PMC2930931  PMID: 19779858
Kainic acid; Rat; Development; Status epilepticus; Stroke; Pentobarbital
13.  Ligand-specific regulation of the extracellular surface of a G protein coupled receptor 
Nature  2010;463(7277):108-112.
G protein coupled receptors (GPCRs) are seven transmembrane proteins that mediate the majority of cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs1,2,3,4,5 reveal structural conservation extending from the orthosteric ligand binding site in the transmembrane core to the cytoplasmic G protein coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse, and therefore represents an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops (ECLs) 2 and 3. Small molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G protein activation (agonist, neutral antagonist, and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide new insight into the dynamic behavior of GPCRs not addressable by static, inactive-state crystal structures.
PMCID: PMC2805469  PMID: 20054398
14.  The Role of Akt/Protein Kinase B Subtypes in Retinal Ischemic Preconditioning 
Experimental eye research  2008;88(3):512-521.
Potent endogenous protection from ischemia can be induced in the retina by ischemic preconditioning (IPC). Protein kinase B/Akt is a cellular survival factor. We hypothesized that Akt was integral to IPC based upon differential effects of Akt subtypes.
Rats were subjected to retinal ischemia after IPC or IPC-mimicking by the opening of mitochondrial KATP (mKATP) channels. The effects of blocking Akt using wortmannin, API-2, or small interfering RNA (siRNA) were examined. Electroretinography assessed functional recovery after ischemia, and TUNEL examined retinal ganglion cell apoptosis. We studied the relationship between Akt activation, and known initiators of IPC, including adenosine receptor stimulation and the opening of mKATP channels.
The PI-3 kinase inhibitor wortmannin 1 or 4 mg/kg (i.p.), the specific Akt inhibitor API-2, 5-500 μM in the vitreous, or intravitreal siRNA directed against Akt2 or -3, but not Akt1, significantly attenuated the neuroprotective effect of IPC. Interfering RNA against any of the three Akt subtypes significantly but time-dependently attenuated mKATP channel opening to mimic IPC. Adenosine A1 receptor blockade (DPCPX), A2a blockade (CSC), or the mKATP channel blocker 5-hydroxydecanoic acid significantly attenuated Akt activation after IPC. Interfering RNA directed against Akt subtypes prevented the ameliorative effect of IPC on post-ischemic apoptosis.
All three Akt subtypes are involved in functional retinal neuroprotection by IPC or IPC-mimicking. Akt is downstream of adenosine A1 and A2a receptors and mKATP channel opening. The results indicate the presence in the retina of robust and redundant endogenous neuroprotection based upon subtypes of Akt.
PMCID: PMC2709455  PMID: 19084003
Akt; electroretinography; ischemic preconditioning; protein kinase B; retinal ischemia
15.  Frequency and Distinctive Spectrum of KRAS Mutations in Never Smokers with Lung Adenocarcinoma 
KRAS mutations are found in ~ 25% of lung adenocarcinomas in Western countries and, as a group, have been strongly associated with cigarette smoking. These mutations are predictive of poor prognosis in resected disease as well as resistance to treatment with erlotinib or gefitinib.
Experimental Design:
We determined the frequency and type of KRAS codon 12 and 13 mutations and characterized their association with cigarette smoking history in patients with lung adenocarcinomas.
KRAS mutational analysis was performed on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12/81; 95% CI 8%-24%) of tumors from never smokers. Similarly, 22% (69/316; 95% CI 17%-27%) of tumors from former smokers, and 25% (21/85; 95% CI 16%-35%) of tumors from current smokers had KRAS mutations. The frequency of KRAS mutation was not associated with age, gender, or smoking history. The number of pack years of cigarette smoking did not predict an increased likelihood of KRAS mutations. Never smokers were significantly more likely than former or current smokers to have a transition mutation (G→A) rather than the transversion mutations known to be smoking related (G→T or G→C; p<0.0001).
Based upon our data, KRAS mutations are not rare among never smokers with lung adenocarcinoma and such patients have a distinct KRAS mutation profile. The etiologic and biological heterogeneity of KRAS mutant lung adenocarcinomas is worthy of further study.
PMCID: PMC2754127  PMID: 18794081
16.  High Resolution Crystal Structure of an Engineered Human β2-Adrenergic G protein-Coupled Receptor 
Science (New York, N.Y.)  2007;318(5854):1258-1265.
G protein-coupled receptors comprise the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human β2-adrenergic receptor—T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 Å resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the β2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.
PMCID: PMC2583103  PMID: 17962520

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