Prefrontal Physiomarkers of Anxiety and Depression in Parkinson's Disease.

Objective: Anxiety and depression are prominent non-motor symptoms of Parkinson's disease (PD), but their pathophysiology remains unclear. We sought to understand their neurophysiological correlates from chronic invasive recordings of the prefrontal cortex (PFC). Methods: We studied four patients undergoing deep brain stimulation (DBS) for their motor signs, who had comorbid mild to moderate anxiety and/or depressive symptoms. In addition to their basal ganglia leads, we placed a permanent prefrontal subdural 4-contact lead. These electrodes were attached to an investigational pulse generator with the capability to sense and store field potential signals, as well as deliver therapeutic neurostimulation. At regular intervals over 3-5 months, participants paired brief invasive neural recordings with self-ratings of symptoms related to depression and anxiety. Results: Mean age was 61 ± 7 years, mean disease duration was 11 ± 8 years and a mean Unified Parkinson's Disease Rating Scale, with part III (UPDRS-III) off medication score of 37 ± 13. Mean Beck Depression Inventory (BDI) score was 14 ± 5 and Beck Anxiety Index was 16.5 ± 5. Prefrontal cortex spectral power in the beta band correlated with patient self-ratings of symptoms of depression and anxiety, with r-values between 0.31 and 0.48. Mood scores showed negative correlation with beta spectral power in lateral locations, and positive correlation with beta spectral power in a mesial recording location, consistent with the dichotomous organization of reward networks in PFC. Interpretation: These findings suggest a physiological basis for anxiety and depression in PD, which may be useful in the development of neurostimulation paradigms for these non-motor disease features.

Prefrontal-Subthalamic Hyperdirect Pathway Modulates Movement Inhibition in Humans.

The ability to dynamically change motor outputs, such as stopping an initiated response, is an important aspect of human behavior. A hyperdirect pathway between the inferior frontal gyrus and subthalamic nucleus is hypothesized to mediate movement inhibition, but there is limited evidence for this in humans. We recorded high spatial and temporal resolution field potentials from both the inferior frontal gyrus and subthalamic nucleus in 21 subjects. Cortical potentials evoked by subthalamic stimulation revealed short latency events indicative of monosynaptic connectivity between the inferior frontal gyrus and ventral subthalamic nucleus. During a stop signal task, stopping-related potentials in the cortex preceded stopping-related activity in the subthalamic nucleus, and synchronization between these task-evoked potentials predicted the stop signal reaction time. Thus, we show that a prefrontal-subthalamic hyperdirect pathway is present in humans and mediates rapid stopping. These findings may inform therapies to treat disorders featuring perturbed movement inhibition.

Deep Analysis of Residue Constraints (DARC): identifying determinants of protein functional specificity.

Protein functional constraints are manifest as superfamily and functional-subgroup conserved residues, and as pairwise correlations. Deep Analysis of Residue Constraints (DARC) aids the visualization of these constraints, characterizes how they correlate with each other and with structure, and estimates statistical significance. This can identify determinants of protein functional specificity, as we illustrate for bacterial DNA clamp loader ATPases. These load ring-shaped sliding clamps onto DNA to keep polymerase attached during replication and contain one δ, three γ, and one δ' AAA+ subunits semi-circularly arranged in the order δ-γ1-γ2-γ3-δ'. Only γ is active, though both γ and δ' functionally influence an adjacent γ subunit. DARC identifies, as functionally-congruent features linking allosterically the ATP, DNA, and clamp binding sites: residues distinctive of γ and of γ/δ' that mutually interact in trans, centered on the catalytic base; several γ/δ'-residues and six γ/δ'-covariant residue pairs within the DNA binding N-termini of helices α2 and α3; and γ/δ'-residues associated with the α2 C-terminus and the clamp-binding loop. Most notable is a trans-acting γ/δ' hydroxyl group that 99% of other AAA+ proteins lack. Mutation of this hydroxyl to a methyl group impedes clamp binding and opening, DNA binding, and ATP hydrolysis-implying a remarkably clamp-loader-specific function.

Washout of chronic therapeutic deep brain stimulation increases cortical phase-amplitude coupling.

Invasive human brain recordings have shown that acute therapeutic deep brain stimulation (DBS) reduces cortical synchronization, measured by coupling of beta phase to gamma amplitude. Here we show by noninvasive scalp electroencephalography that withdrawal of chronic DBS elevates phase-amplitude coupling, in proportion to the worsening of contralateral rigidity.

Altered Prefrontal Theta and Gamma Activity during an Emotional Face Processing Task in Parkinson Disease.

Patients with Parkinson disease (PD) often experience nonmotor symptoms including cognitive deficits, depression, and anxiety. Cognitive and affective processes are thought to be mediated by prefrontal cortico-basal ganglia circuitry. However, the topography and neurophysiology of prefrontal cortical activity during complex tasks are not well characterized. We used high-resolution electrocorticography in pFC of patients with PD and essential tremor, during implantation of deep brain stimulator leads in the awake state, to understand disease-specific changes in prefrontal activity during an emotional face processing task. We found that patients with PD had less task-related theta-alpha power and greater task-related gamma power in the dorsolateral pFC, inferior frontal cortex, and lateral OFC. These findings support a model of prefrontal neurophysiological changes in the dopamine-depleted state, in which focal areas of hyperactivity in prefrontal cortical regions may compensate for impaired long-range interactions mediated by low-frequency rhythms. These distinct neurophysiological changes suggest that nonmotor circuits undergo characteristic changes in PD.

Effect of levodopa on electroencephalographic biomarkers of the parkinsonian state.

The objective of this study was to evaluate proposed electroencephalographic (EEG) biomarkers of Parkinson's disease (PD) and test their correlation with motor impairment in a new, well-characterized cohort of PD patients and controls. Sixty-four-channel EEG was recorded from 14 patients with rigid-akinetic PD with minimal tremor and from 14 age-matched healthy controls at rest and during voluntary movement. Patients were tested off and on medication during a single session. Recordings were analyzed for phase-amplitude coupling over sensorimotor cortex and for pairwise coherence from all electrode pairs in the recording montage (distributed coherence). Phase-amplitude coupling and distributed coherence were found to be elevated Off compared with On levodopa, and their reduction was correlated with motor improvement. In the Off medication state, phase-amplitude coupling was greater in sensorimotor contacts contralateral to the most affected body part and reduced by voluntary movement. We conclude that phase-amplitude coupling and distributed coherence are cortical biomarkers of the parkinsonian state that are detectable noninvasively and may be useful as objective aids for management of dopaminergic therapy. Several analytic methods may be used for noninvasive measurement of abnormal brain synchronization in PD. Calculation of phase-amplitude coupling requires only a single electrode over motor cortex. NEW & NOTEWORTHY Several EEG biomarkers of the parkinsonian state have been proposed that are related to abnormal cortical synchronization. We report several new findings in this study: correlations of EEG markers of synchronization with specific motor signs of Parkinson's disease (PD), and demonstration that one of the EEG markers, phase-amplitude coupling, is more elevated over the more clinically affected brain hemisphere. These findings underscore the potential utility of scalp EEG for objective, noninvasive monitoring of medication state in PD.

Adaptive deep brain stimulation for Parkinson's disease using motor cortex sensing.

OBJECTIVE: Contemporary deep brain stimulation (DBS) for Parkinson's disease is delivered continuously, and adjustments based on patient's changing symptoms must be made manually by a trained clinician. Patients may be subjected to energy intensive settings at times when they are not needed, possibly resulting in stimulation-induced adverse effects, such as dyskinesia. One solution is 'adaptive' DBS, in which stimulation is modified in real time based on neural signals that co-vary with the severity of motor signs or of stimulation-induced adverse effects. Here we show the feasibility of adaptive DBS using a fully implanted neural prosthesis. APPROACH: We demonstrate adaptive deep brain stimulation in two patients with Parkinson's disease using a fully implanted neural prosthesis that is enabled to utilize brain sensing to control stimulation amplitude (Activa PC + S). We used a cortical narrowband gamma (60-90 Hz) oscillation related to dyskinesia to decrease stimulation voltage when gamma oscillatory activity is high (indicating dyskinesia) and increase stimulation voltage when it is low. MAIN RESULTS: We demonstrate the feasibility of 'adaptive deep brain stimulation' in two patients with Parkinson's disease. In short term in-clinic testing, energy savings were substantial (38%-45%), and therapeutic efficacy was maintained. SIGNIFICANCE: This is the first demonstration of adaptive DBS in Parkinson's disease using a fully implanted device and neural sensing. Our approach is distinct from other strategies utilizing basal ganglia signals for feedback control.

Subthalamic local field potentials in Parkinson's disease and isolated dystonia: An evaluation of potential biomarkers.

Local field potentials (LFP) recorded from the subthalamic nucleus in patients with Parkinson's disease (PD) demonstrate prominent oscillations in the beta (13-30 Hz) frequency range, and reduction of beta band spectral power by levodopa and deep brain stimulation (DBS) is correlated with motor symptom improvement. Several features of beta activity have been theorized to be specific biomarkers of the parkinsonian state, though these have rarely been studied in non-parkinsonian conditions. To compare resting state LFP features in PD and isolated dystonia and evaluate disease-specific biomarkers, we recorded subthalamic LFPs from 28 akinetic-rigid PD and 12 isolated dystonia patients during awake DBS implantation. Spectral power and phase-amplitude coupling characteristics were analyzed. In 26/28 PD and 11/12 isolated dystonia patients, the LFP power spectrum had a peak in the beta frequency range, with similar amplitudes between groups. Resting state power did not differ between groups in the theta (5-8 Hz), alpha (8-12 Hz), beta (13-30 Hz), broadband gamma (50-200 Hz), or high frequency oscillation (HFO, 250-350 Hz) bands. Analysis of phase-amplitude coupling between low frequency phase and HFO amplitude revealed significant interactions in 19/28 PD and 6/12 dystonia recordings without significant differences in maximal coupling or preferred phase. Two features of subthalamic LFPs that have been proposed as specific parkinsonian biomarkers, beta power and coupling of beta phase to HFO amplitude, were also present in isolated dystonia, including focal dystonias. This casts doubt on the utility of these metrics as disease-specific diagnostic biomarkers.

Inflammation-associated interleukin-6/signal transducer and activator of transcription 3 activation ameliorates alcoholic and nonalcoholic fatty liver diseases in interleukin-10-deficient mice.

UNLABELLED: Alcoholic and nonalcoholic steatohepatitis are characterized by fatty liver plus inflammation. It is generally believed that steatosis promotes inflammation, whereas inflammation in turn aggregates steatosis. Thus, we hypothesized the deletion of interleukin (IL)-10, a key anti-inflammatory cytokine, exacerbates liver inflammation, steatosis, and hepatocellular damage in alcoholic and nonalcoholic fatty liver disease models that were achieved via feeding mice with a liquid diet containing 5% ethanol for 4 weeks or a high-fat diet (HFD) for 12 weeks, respectively. IL-10 knockout (IL-10(-/-)) mice and several other strains of genetically modified mice were generated and used. Compared with wild-type mice, IL-10(-/-) mice had greater liver inflammatory response with higher levels of IL-6 and hepatic signal transducer and activator of transcription 3 (STAT3) activation, but less steatosis and hepatocellular damage after alcohol or HFD feeding. An additional deletion of IL-6 or hepatic STAT3 restored steatosis and hepatocellular damage but further enhanced liver inflammatory response in IL-10(-/-) mice. In addition, the hepatic expression of sterol regulatory element-binding protein 1 and key downstream lipogenic proteins and enzymes in fatty acid synthesis were down-regulated in IL-10(-/-) mice. Conversely, IL-10(-/-) mice displayed enhanced levels of phosphorylated adenosine monophosphate-activated protein kinase and its downstream targets including phosphorylated acetyl-coenzyme A carboxylase and carnitine palmitoyltransferase 1 in the liver. Such dysregulations were corrected in IL-10(-/-) IL-6(-/-) or IL-10(-/-) STAT3(Hep-/-) double knockout mice. CONCLUSION: IL-10(-/-) mice are prone to liver inflammatory response but are resistant to steatosis and hepatocellular damage induced by ethanol or HFD feeding. Resistance to steatosis in these mice is attributable to elevation of inflammation-associated hepatic IL-6/STAT3 activation that subsequently down-regulates lipogenic genes but up-regulates fatty acid oxidation-associated genes in the liver.

Fire regime in a Mexican forest under indigenous resource management.

The Rarámuri (Tarahumara) people live in the mountains and canyons of the Sierra Madre Occidental of Chihuahua, Mexico. They base their subsistence on multiple-use strategies of their natural resources, including agriculture, pastoralism, and harvesting of native plants and wildlife. Pino Gordo is a Rarámuri settlement in a remote location where the forest has not been commercially logged. We reconstructed the forest fire regime from fire-scarred trees, measured the structure of the never-logged forest, and interviewed community members about fire use. Fire occurrence was consistent throughout the 19th and 20th centuries up to our fire scar collection in 2004. This is the least interrupted surface-fire regime reported to date in North America. Studies from other relict sites such as nature reserves in Mexico or the USA have all shown some recent alterations associated with industrialized society. At Pino Gordo, fires recurred frequently at the three study sites, with a composite mean fire interval of 1.9 years (all fires) to 7.6 years (fires scarring 25% or more of samples). Per-sample fire intervals averaged 10-14 years at the three sites. Approximately two-thirds of fires burned in the season of cambial dormancy, probably during the pre-monsoonal drought. Forests were dominated by pines and contained many large living trees and snags, in contrast to two nearby similar forests that have been logged. Community residents reported using fire for many purposes, consistent with previous literature on fire use by indigenous people. Pino Gordo is a valuable example of a continuing frequent-fire regime in a never-harvested forest. The Rarámuri people have actively conserved this forest through their traditional livelihood and management techniques, as opposed to logging the forest, and have also facilitated the fire regime by burning. The data contribute to a better understanding of the interactions of humans who live in pine forests and the fire regimes of these ecosystems, a topic that has been controversial and difficult to assess from historical or paleoecological evidence.

Hepatoprotective versus oncogenic functions of STAT3 in liver tumorigenesis.

Aberrantly hyperactivated STAT3 has been found in human liver cancers as an oncogene; however, STAT3 has also been shown to exert hepatoprotective effects during liver injury. The balancing act that STAT3 plays between hepatoprotection and liver tumorigenesis remains poorly defined. In this study, the diethylnitrosamine (DEN)-induced liver tumor model and the chronic carbon tetrachloride (CCl(4))-induced liver fibrosis model were both used to investigate the role of STAT3 in liver tumorigenesis. Hepatocyte-specific STAT3 knockout mice were resistant to liver tumorigenesis induced by a single DEN injection, whose tumorigenesis was associated with minimal chronic liver inflammation, injury, and fibrosis. In contrast, long-term CCl(4) treatment resulted in severe hepatic oxidative damage, inflammation, and fibrosis but rarely induced liver tumor formation in wild-type mice. Despite the oncogenic function of STAT3 in DEN-induced liver tumor, hepatocyte-specific STAT3 knockout mice were more susceptible to liver tumorigenesis after 16 weeks of CCl(4) injection, which was associated with higher levels of liver injury, inflammation, fibrosis, and oxidative DNA damage compared with wild-type mice. These findings suggest that the hepatoprotective feature of STAT3 prevents hepatic damage and fibrosis under the condition of persistent inflammatory stress, consequently suppressing injury-driven liver tumor initiation. Once liver tumor cells have developed, STAT3 likely acts as an oncogenic factor to promote tumor growth.

Suppression of innate immunity (natural killer cell/interferon-γ) in the advanced stages of liver fibrosis in mice.

UNLABELLED: Activation of innate immunity (natural killer [NK] cell/interferon-γ [IFN-γ]) has been shown to play an important role in antiviral and antitumor defenses as well as antifibrogenesis. However, little is known about the regulation of innate immunity during chronic liver injury. Here, we compared the functions of NK cells in early and advanced liver fibrosis induced by a 2-week or a 10-week carbon tetrachloride (CCl(4) ) challenge, respectively. Injection of polyinosinic-polycytidylic acid (poly I:C) or IFN-γ induced NK cell activation and NK cell killing of hepatic stellate cells (HSCs) in the 2-week CCl(4) model. Such activation was diminished in the 10-week CCl(4) model. Consistent with these findings, the inhibitory effect of poly I:C and IFN-γ on liver fibrosis was markedly reduced in the 10-week versus the 2-week CCl(4) model. In vitro coculture experiments demonstrated that 4-day cultured (early activated) HSCs induce NK cell activation via an NK group 2 member D/retinoic acid-induced early gene 1-dependent mechanism. Such activation was reduced when cocultured with 8-day cultured (intermediately activated) HSCs due to the production of transforming growth factor-ß (TGF-ß) by HSCs. Moreover, early activated HSCs were sensitive, whereas intermediately activated HSCs were resistant to IFN-γ-mediated inhibition of cell proliferation, likely due to elevated expression of suppressor of cytokine signaling 1 (SOCS1). Disruption of the SOCS1 gene restored the IFN-γ inhibition of cell proliferation in intermediately activated HSCs. Production of retinol metabolites by HSCs contributed to SOCS1 induction and subsequently inhibited IFN-γ signaling and functioning, whereas production of TGF-ß by HSCs inhibited NK cell function and cytotoxicity against HSCs. CONCLUSION: The antifibrogenic effects of NK cell/IFN-γ are suppressed during advanced liver injury, which is likely due to increased production of TGF-ß and expression of SOCS1 in intermediately activated HSCs.

Molecular mechanisms of alcoholic liver disease: innate immunity and cytokines.

Alcohol consumption is a predominant etiological factor in the pathogenesis of chronic liver diseases worldwide, causing fatty liver, alcoholic hepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma. In the past few decades, significant progress has been made in our understanding of the molecular mechanisms underlying alcoholic liver injury. Activation of innate immunity components such as Kupffer cells, LPS/TLR4, and complements in response to alcohol exposure plays a key role in the development and progression of alcoholic liver disease (ALD). LPS activation of Kupffer cells also produces IL-6 and IL-10 that may play a protective role in ameliorating ALD. IL-6 activates signal transducer and activator of transcription 3 (STAT3) in hepatocytes and sinusoidal endothelial cells, while IL-10 activates STAT3 in Kupffer cells/macrophages, subsequently protecting against ALD. In addition, alcohol consumption also inhibits some components of innate immunity such as natural killer (NK) cells, a type of cells that play key roles in anti-viral, anti-tumor, and anti-fibrotic defenses in the liver. Ethanol inhibition of NK cells likely contributes significantly to the pathogenesis of ALD. Understanding the roles of innate immunity and cytokines in alcoholic liver injury may provide insight into novel therapeutic targets in the treatment of alcoholic liver disease.

Th17 cells and their associated cytokines in liver diseases.

T helper 17 (Th17) cells are a newly identified subset of T helper cells that play important roles in host defense against extracellular bacteria as well as in the pathogenesis of autoimmune disease. The functions of Th17 cells are mediated via the production of several cytokines including interleukin (IL)-17 and IL-22. Recent studies show that the frequency of IL-17(+) cells is significantly elevated in a variety of chronic liver diseases including alcoholic liver disease, viral hepatitis and hepatocellular carcinoma. IL-17 receptor is expressed virtually on all types of liver cells, while IL-22 receptor expression is restricted to epithelial cells including hepatocytes in the liver. IL-17 seems to play an important role in inducing liver inflammation via stimulating multiple types of liver nonparenchymal cells to produce proinflammatory cytokines and chemokines, while IL-22 appears to be an important factor in promoting hepatocyte survival and proliferation.

Dissociation between liver inflammation and hepatocellular damage induced by carbon tetrachloride in myeloid cell-specific signal transducer and activator of transcription 3 gene knockout mice.

UNLABELLED: Liver injury is associated with inflammation, which is generally believed to accelerate the progression of liver diseases; however, clinical data show that inflammation does not always correlate with hepatocelluar damage in some patients. Investigating the cellular mechanisms underlying these events using an experimental animal model, we show that inflammation may attenuate liver necrosis induced by carbon tetrachloride (CCl(4)) in myeloid-specific signal transducer and activator of transcription 3 (STAT3) knockout mice. As an important anti-inflammatory signal, conditional deletion of STAT3 in myeloid cells results in markedly enhanced liver inflammation after CCl(4) injection. However, these effects are also accompanied by reduced liver necrosis, correlating with elevated serum interleukin-6 (IL-6) and hepatic STAT3 activation. An additional deletion of STAT3 in hepatocytes in myeloid-specific STAT3 knockout mice restored hepatic necrosis but decreased liver inflammation. CONCLUSION: Inflammation-mediated STAT3 activation attenuates hepatocellular injury induced by CCl(4) in myeloid-specific STAT3 knockout mice, suggesting that inflammation associated with a predominance of hepatoprotective cytokines that activate hepatic STAT3 may reduce rather than accelerate hepatocellular damage in patients with chronic liver diseases.

Anti-inflammatory and anti-apoptotic roles of endothelial cell STAT3 in alcoholic liver injury.

BACKGROUND: It is generally believed that the hepatoprotective effect of interleukin-6 (IL-6) is mediated via activation of signal transducer and activator of transcription 3 (STAT3) in hepatocytes. IL-6-deficient mice are more susceptible to alcohol-induced hepatocyte apoptosis and steatosis and elevation of serum alanine transaminase (ALT); however, whereas hepatocyte-specific STAT3 knockout mice are more susceptible to alcohol-induced hepatic steatosis, they have similar hepatocyte apoptosis and serum ALT after alcohol feeding compared with wild-type mice. This suggests that the hepatoprotective effect of IL-6 in alcoholic liver injury may be mediated via activation of STAT3-independent signals in hepatocytes, activation of STAT3 in nonparenchymal cells, or both. We have previously shown that IL-6 also activates STAT3 in sinusoidal endothelial cells (SECs). Thus, the purpose of this study was to investigate whether STAT3 in endothelial cells also plays a protective role in alcoholic liver injury. METHODS: Wild-type and endothelial cell-specific STAT3 knockout (STAT3(E-/-)) mice were pair-fed and fed ethanol containing diet for 4 weeks. Liver injury and inflammation were determined. RESULTS: Feeding mice with ethanol-containing diet for 4 weeks induced greater hepatic injury (elevation of serum ALT) and liver weight in STAT3(E-/-) mice than wild-type control groups. In addition, ethanol-fed STAT3(E-/-) mice displayed greater hepatic inflammation and substantially elevated serum and hepatic levels of IL-6 and TNF-alpha compared with wild-type mice. Furthermore, ethanol-fed STAT3(E-/-) mice displayed a greater abundance of apoptotic SECs and higher levels of serum hyaluronic acid than wild-type controls. CONCLUSIONS: These data suggest that endothelial cell STAT3 plays important dual functions of attenuating hepatic inflammation and SEC death during alcoholic liver injury.

Altered endothelin-1 signaling in production of thromboxane A2 in kupffer cells from bile duct ligated rats.

Kupffer cells (KCs), the liver resident macrophages accounting for 80-90% of the total population of fixed tissue macrophages in the body, not only play a key role in host defense via removing particulate materials from the portal circulation, but may also contribute to the pathogenesis of various liver diseases. We have previously demonstrated that KCs play an important role in controlling portal hypertension and hepatocellular injury via releasing thromboxane A2 (TXA2) in early fibrosis induced by one-week bile duct ligation (BDL). Production of TXA2 is controlled by cytosolic phospholipase A2 (cPLA2) that is activated by the interaction of entothelin-1 (ET-1) with its G-protein coupled ET receptor B (ETBR). However, the signaling pathways that contribute to the ET-1-induced activation of cPLA2 and production of TXA2 in KCs in the normal healthy or injured livers are not yet clear, which are investigated in the present study using isolated KCs from one-week BDL or sham rats. The pharmacological inhibition of cPLA2 or chelation of intracellular calcium abrogated the ET-1 induction of TXA2 from KCs. Compared to those from sham rats, KCs from BDL animals displayed a significantly enhanced responsiveness of p38 MAPK to ET-1, increased ETBR and Galphai subunit but decreased Galphaq and Galpha11 expression. Inhibition of ERK1/2 or Gq signaling abrogated significantly the ET-1 induction of TXA2 in sham KCs but only slightly in BDL KCs. In contrast, inhibition of p38 MAPK and Gi signaling markedly attenuated the ET-1 induction of TXA2 in BDL KCs but had no effect in sham KCs. Lastly, inhibition of PLC or PKC abrogated ET-1 induction of TXA2 in KCs from both sham and BDL groups. The hepatic stress (such as BDL) induces significant modifications in the receptor and intermediates of ET-1 signaling in KC and subsequently alters ET-1 signaling mechanisms, particularly a shift from Gq induced signaling to Gi induced signaling, in the activation of cPLA2 and production of TXA2 in response to ET-1.

Interpretation of p-cyanophenylalanine fluorescence in proteins in terms of solvent exposure and contribution of side-chain quenchers: a combined fluorescence, IR and molecular dynamics study.

The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.

Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan.

The experimental study of protein folding is enhanced by the use of nonintrusive probes that are sensitive to local conformational changes in the protein structure. Here, we report the selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that can function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding. l-4-Cyanophenylalanine (pCNPhe) and 4-ethynylphenylalanine (pENPhe) were incorporated into the hydrophobic core of the 171-residue protein, T4 lysozyme. The FRET donor ability of pCNPhe and pENPhe is evident by the overlap of the emission spectra of pCNPhe and pENPhe with the absorbance spectrum of Trp. The incorporation of both unnatural amino acids in place of a phenylalanine in the hydrophobic core of T4 lysozyme was well tolerated by the protein, due in part to the small size of the cyano and ethynyl groups. The hydrophobic nature of the ethynyl group of pENPhe suggests that this unnatural amino acid is a more conservative substitution into the hydrophobic core of the protein compared to pCNPhe. The urea-induced disruption of the hydrophobic core of the protein was probed by the change in FRET efficiency between either pCNPhe or pENPhe and the Trp residues in T4 lysozyme. The methodology for the study of protein conformational changes using FRET presented here is of general applicability to the study of protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.

Rapamycin does not improve insulin sensitivity despite elevated mammalian target of rapamycin complex 1 activity in muscles of ob/ob mice.

Studies of cultured cells have indicated that the mammalian target of rapamycin complex 1 (mTORC1) mediates the development of insulin resistance. Because a role for mTORC1 in the development of skeletal muscle insulin resistance has not been established, we studied mTORC1 activity in skeletal muscles of ob/ob (OB) mice and wild-type (WT) mice. In vivo insulin action was assessed in muscles of mice 15 min following an intraperitoneal injection of insulin or an equivalent volume of saline. In the basal state, the phosphorylation of S6K on Thr(389), mTOR on Ser(2448), and PRAS40 on Thr(246) were increased significantly in muscles from OB mice compared with WT mice. The increase in basal mTORC1 signaling was associated with an increase in basal PKB phosphorylation on Thr(308) and Ser(473). In the insulin-stimulated state, no differences existed in the phosphorylation of S6K on Thr(389), but PKB phosphorylation on Thr(308) and Ser(473) was significantly reduced in muscles of OB compared with WT mice. Despite elevated mTORC1 activity in OB mice, rapamycin treatment did not improve either glucose tolerance or insulin tolerance. These results indicate that the insulin resistance of OB mice is mediated, in part, by factors other than mTORC1.