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Abstract The binding of insulin to the insulin receptor (IR) triggers a cascade of receptor conformational changes and autophosphorylation, leading to the activation of metabolic and mitogenic pathways. Recent advances in the structural and functional analyses of IR have revealed the conformations of the extracellular domains of the IR in inactive and fully activated states. However, the early activation mechanisms of this receptor remain poorly understood. The structures of partially activated IR in complex with aptamers provide clues for understanding the initial activation mechanism. In this review, we discuss the structural and functional features of IR complexed with various ligands and propose a model to explain the sequential activation mechanism. Moreover, we discuss the structures of IR complexed with biased agonists that selectively activate metabolic pathways and provide insights into the design of selective agonists and their clinical implications.

Aminoacyl-tRNA synthetases possess unique domains. In this study the structure of the vertebrate IARS1 and EARS1 complex reveals that vertebrate IARS1 protects the DNA repair factor BRCA1 from proteolytic degradation via its UBX-fold domain.

DNA aptamers can activate insulin receptor as selective agonists or positive allosteric modulators. Here, authors determine structures of the insulin receptor bound to aptamers and provide a basis for the selective activation or allosteric regulation of insulin receptor.

Gut health: intestinal barrier function underpins health and disease A better understanding of how the cells that line the inside of the intestines allow nutrients in, while keeping harmful substances and pathogens out could lead to new therapies for inflammatory bowel disease, obesity, and other conditions. A team from South Korea led by Sung Ho Ryu from Pohang University of Science and Technology review the regulatory mechanisms that help maintain the intestinal epithelial barrier. They discuss the role of tight junction proteins in forming a seal between adjacent cells and the various signaling pathways that loosen or tighten these junctions to enable limited transport. Loss of barrier integrity because of genetics, gut microbes, auto-immunity, diet, or other factors is often implicated in disease, and restoring barrier function with drugs or probiotics could help ameliorate many health problems.

Cancer: secreted proteins associated with fat-induced tumor growth By exposing liver cancer cells to oleate, an unsaturated fatty acid, researchers have discovered a group of secreted proteins that may help explain why fatty acids increase proliferative capacity in tumors. Soyeon Park from Pohang University of Science and Technology in South Korea and coworkers treated liver cancer cells with oleate and then measured all the proteins released from the cells. Comparison with untreated cells revealed 145 proteins secreted at elevated levels—most of which were involved in metabolism, stress responses and other proliferation-related processes—and another 192 proteins secreted at reduced levels. The researchers ran additional biochemical analyses on six secreted proteins to validate the changes following exposure to oleate. The authors suggest that these validated proteins could now serve as biomarkers of tumor aggressiveness or as future drug targets.

Abstract Robust mitochondrial respiration provides energy to support physical performance and physiological well-being, whereas mitochondrial malfunction is associated with various pathologies and reduced longevity. In the current study, we tested whether myricetin, a natural flavonol with diverse biological activities, may impact mitochondrial function and longevity. The mice were orally administered myricetin (50 mg/kg/day) for 3 weeks. Myricetin significantly potentiated aerobic capacity in mice, as evidenced by their increased running time and distance. The elevated mitochondrial function was associated with induction of genes for oxidative phosphorylation and mitochondrial biogenesis in metabolically active tissues. Importantly, myricetin treatment led to decreased PGC-1α acetylation through SIRT1 activation. Furthermore, myricetin significantly improved the healthspan and lifespan of wild-type, but not Sir-2.1-deficient, C. elegans. These results demonstrate that myricetin enhances mitochondrial activity, possibly by activating PGC-1α and SIRT1, to improve physical endurance, strongly suggesting myricetin as a mitochondria-activating agent.

Abstract Ulcerative colitis is a multi-factorial disease involving a dysregulated immune response. Disruptions to the intestinal epithelial barrier and translocation of bacteria, resulting in inflammation, are common in colitis. The mechanisms underlying epithelial barrier dysfunction or regulation of tight junction proteins during disease progression of colitis have not been clearly elucidated. Increase in phospholipase D (PLD) activity is associated with disease severity in colitis animal models. However, the role of PLD2 in the maintenance of intestinal barrier integrity remains elusive. We have generated intestinal-specific Pld2 knockout mice (Pld2 IEC-KO) to investigate the mechanism of intestinal epithelial PLD2 in colitis. We show that the knockout of Pld2 confers protection against dextran sodium sulphate (DSS)-induced colitis in mice. Treatment with DSS induced the expression of PLD2 and downregulated occludin in colon epithelial cells. PLD2 was shown to mediate phosphorylation of occludin and induce its proteasomal degradation in a c-Src kinase-dependent pathway. Additionally, we have shown that treatment with an inhibitor of PLD2 can rescue mice from DSS-induced colitis. To our knowledge, this is the first report showing that PLD2 is pivotal in the regulation of the integrity of epithelial tight junctions and occludin turn over, thereby implicating it in the pathogenesis of colitis.

IL-17 produced by Th17 cells has been implicated in the pathogenesis of rheumatoid arthritis (RA). It is important to prevent the differentiation of Th17 cells in RA. Homodimeric soluble γc (sγc) impairs IL-2 signaling and enhances Th17 differentiation. Thus, we aimed to block the functions of sγc by inhibiting the formation of homodimeric sγc. The homodimeric form of sγc was strikingly disturbed by sγc-binding DNA aptamer. Moreover, the aptamer effectively inhibited Th17 cell differentiation and restored IL-2 and IL-15 signaling impaired by sγc with evidences of increased survival of T cells. sγc was highly expressed in SF of RA patients and increased in established CIA mice. The therapeutic effect of PEG-aptamer was tested in CIA model and its treatment alleviated arthritis pathogenesis with impaired differentiation of pathogenic Th17, NKT1, and NKT17 cells in inflamed joint. Homodimeric sγc has pathogenic roles to exacerbate RA progression with differentiation of local Th17, NKT1, and NKT17 cells. Therefore, sγc is suggested as target of a therapeutic strategy for RA.

Current methods to measure post-translational modifications of receptor tyrosine kinases provide ensemble results. Here the authors develop a single-molecule blotting (SiMBlot) assay that detects site-specific phosphorylation patterns at the single-molecule level.

Interactions between membrane proteins are poorly understood despite their importance in cell signaling and drug development. Here, we present a co-immunoimmobilization assay (Co-II) enabling the direct observation of membrane protein interactions in single living cells that overcomes the limitations of currently prevalent proximity-based indirect methods. Using Co-II, we investigated the transient homodimerizations of epidermal growth factor receptor (EGFR) and beta-2 adrenergic receptor (β2-AR) in living cells, revealing the differential regulation of these receptors' dimerizations by molecular conformations and microenvironment in a plasma membrane. Co-II should provide a simple, rapid, and robust platform for visualizing both weak and strong protein interactions in the plasma membrane of living cells.

Insulin resistance is a syndrome that affects multiple insulin target tissues, each having different biological functions regulated by insulin. A remaining question is to mechanistically explain how an insulin target cell/tissue can be insulin resistant in one biological function and insulin sensitive in another at the same time. Here, we provide evidence that in pancreatic β cells, knockdown of PI3K-C2α expression results in rerouting of the insulin signal from insulin receptor (IR)-B/PI3K-C2α/PKB-mediated metabolic signaling to IR-B/Shc/ERK-mediated mitogenic signaling, which allows the β cell to switch from a highly glucose-responsive, differentiated state to a proliferative state. Our data suggest the existence of IR-cascade-selective insulin resistance, which allows rerouting of the insulin signal within the same target cell. Hence, factors involved in the rerouting of the insulin signal represent tentative therapeutic targets in the treatment of insulin resistance.

Insulin is secreted in a pulsatile manner from multiple micro-organs called the islets of Langerhans. The amplitude and phase (shape) of insulin secretion are modulated by numerous factors including glucose. The role of phase modulation in glucose homeostasis is not well understood compared to the obvious contribution of amplitude modulation. In the present study, we measured Ca2+ oscillations in islets as a proxy for insulin pulses, and we observed their frequency and shape changes under constant/alternating glucose stimuli. Here we asked how the phase modulation of insulin pulses contributes to glucose regulation. To directly answer this question, we developed a phenomenological oscillator model that drastically simplifies insulin secretion, but precisely incorporates the observed phase modulation of insulin pulses in response to glucose stimuli. Then, we mathematically modeled how insulin pulses regulate the glucose concentration in the body. The model of insulin oscillation and glucose regulation describes the glucose-insulin feedback loop. The data-based model demonstrates that the existence of phase modulation narrows the range within which the glucose concentration is maintained through the suppression/enhancement of insulin secretion in conjunction with the amplitude modulation of this secretion. The phase modulation is the response of islets to glucose perturbations. When multiple islets are exposed to the same glucose stimuli, they can be entrained to generate synchronous insulin pulses. Thus, we conclude that the phase modulation of insulin pulses is essential for glucose regulation and inter-islet synchronization.

Pancreatic islets can adapt to oscillatory glucose to produce synchronous insulin pulses. Can islets adapt to other oscillatory stimuli, specifically insulin? To answer this question, we stimulated islets with pulses of exogenous insulin and measured their Ca2+ oscillations. We observed that sufficiently high insulin (> 500 nM) with an optimal pulse period (~ 4 min) could make islets to produce synchronous Ca2+ oscillations. Glucose and insulin, which are key stimulatory factors of islets, modulate islet Ca2+ oscillations differently. Glucose increases the active-to-silent ratio of phases, whereas insulin increases the period of the oscillation. To examine the dual modulation, we adopted a phase oscillator model that incorporated the phase and frequency modulations. This mathematical model showed that out-of-phase oscillations of glucose and insulin were more effective at synchronizing islet Ca2+ oscillations than in-phase stimuli. This finding suggests that a phase shift in glucose and insulin oscillations can enhance inter-islet synchronization.

Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue.

Endothelial progenitor cells (EPCs) can be isolated from human bone marrow or peripheral blood and reportedly contribute to neovascularization. Aptamers are 40-120-mer nucleotides that bind to a specific target molecule, as antibodies do. To utilize apatmers for isolation of EPCs, in the present study, we successfully generated aptamers that recognize human CD31, an endothelial cell marker. CD31 aptamers bound to human umbilical cord blood-derived EPCs and showed specific interaction with human CD31, but not with mouse CD31. However, CD31 aptamers showed non-specific interaction with CD31-negative 293FT cells and addition of polyanionic competitor dextran sulfate eliminated non-specific interaction without affecting cell viability. From the mixture of EPCs and 293FT cells, CD31 aptamers successfully isolated EPCs with 97.6% purity and 94.2% yield, comparable to those from antibody isolation. In addition, isolated EPCs were decoupled from CD31 aptamers with a brief treatment of high concentration dextran sulfate. EPCs isolated with CD31 aptamers and subsequently decoupled from CD31 aptamers were functional and enhanced the restoration of blood flow when transplanted into a murine hindlimb ischemia model. In this study, we demonstrated isolation of foreign material-free EPCs, which can be utilized as a universal protocol in preparation of cells for therapeutic transplantation.

We developed a process to produce novel interactions between two previously unrelated proteins. This process selects protein scaffolds and designs protein interfaces that bind to a surface patch of interest on a target protein. Scaffolds with shapes complementary to the target surface patch were screened using an exhaustive computational search of the human proteome and optimized by directed evolution using phage display. This method was applied to successfully design scaffolds that bind to epidermal growth factor receptor (EGFR) domain II, the interface of EGFR dimerization, with high reactivity toward the target surface patch of EGFR domain II. One potential application of these tailor-made protein interactions is the development of therapeutic agents against specific protein targets.

5' AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl)-thioureido]-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.

Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated.Here, we found that AMPK-induced phospholipase D1 (PLD1) activation is required for (14)C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT) stimulates PLD activity, while AMPK-dominant negative (DN) inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in (14)C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of (14)C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK) is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA), which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate (14)C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator) regulate (14)C-glucose uptake and cell surface glucose transport (GLUT) 4 through ERK stimulation by AMPK-mediated PLD1 activation.These results suggest that AMPK-mediated PLD1 activation is required for (14)C-glucose uptake through ERK stimulation. We propose that the AMPK-mediated PLD1 pathway may provide crucial clues to understanding the mechanisms involved in glucose uptake.

BACKGROUND:Upon ligand binding, cell surface signaling receptors are internalized through a process tightly regulated by endocytic proteins and adaptor protein 2 (AP2) to orchestrate them. Although the molecular identities and roles of endocytic proteins are becoming clearer, it is still unclear what determines the receptor endocytosis kinetics which is mainly regulated by the accumulation of endocytic apparatus to the activated receptors. METHODOLOGY/PRINCIPAL FINDINGS:Here we employed the kinetic analysis of endocytosis and adaptor recruitment to show that mu2, a subunit of AP2 interacts directly with phospholipase D (PLD)1, a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). We also demonstrate that the PLD1-mu2 interaction requires the binding of PLD1 with phosphatidic acid, its own product. CONCLUSIONS/SIGNIFICANCE:These results suggest that the temporal regulation of EGFR endocytosis is achieved by auto-regulatory PLD1 which senses the receptor activation and triggers the translocation of AP2 near to the activated receptor.

Research has been conducted on the mammalian phospholipase D (PLD) and its isozymes, PLD1 and PLD2. The regulatory mechanisms of PLD2 have been investigated and the results suggest that one of the glycolytic enzymes, aldolase A regulates PLD2 activity depending on its metabolite availability.

Researchers investigated the functions in the brain of two of the four isoenzymes of the enzyme phospholipase C (PLC). They studied the functions of PLC-beta1 and PLC-beta4 by generating null mutants in mice. The PLC-beta1 mice developed epilepsy and the PLC-beta4 mice demonstrated ataxia. The research revealed that PLC-beta1 affects signal transduction in the hippocampus and cerebral cortex, and that PLC-beta4 affects the metabotropic glutamate receptor in the cerebellum. These results indicate that neurological disorders have a pharmacological basis.

Patients with amyotrophic lateral sclerosis (ALS) often show hallmarks of type 2 diabetes mellitus (T2DM). However, the causal link between ALS and T2DM has remained a mystery. We now demonstrate that 60% of ALS patients with T2DM (ALS-T2DM) have sera that exaggerated K+-induced increases in cytosolic free Ca2+ concentration ([Ca2+]i) in mouse islet cells. The effect was attributed to the presence of pathogenic immunoglobulin Gs (IgGs) in ALST2DM sera. The pathogenic IgGs immunocaptured the voltagedependent Ca2+ (CaV) channel subunit CaVα2δ1 in the plasmamembrane enhancing CaV1 channel-mediated Ca2+ influx and [Ca2+]i, resulting in impaired mitochondrial function. Consequently, impairments in [Ca2+]i dynamics, insulin secretion, and cell viability occurred. These data reveal that patients with ALS-T2DM carry cytotoxic ALS-T2DM-IgG autoantibodies that serve as a causal link between ALS and T2DM by immunoattacking CaαV2δ1 subunits. Our findings may lay the foundation for a pharmacological treatment strategy for patients suffering froma combination of these diseases. [ABSTRACT FROM AUTHOR]

Adipose tissue functions as an endocrine organ, and the development of systemic inflammation in adipose tissue is closely associated with metabolic diseases, such as obesity and insulin resistance. Accordingly, the fine regulation of the inflammatory response caused by obesity has therapeutic potential for the treatment of metabolic syndrome. In this study, we analyzed the role of DJ-1 (PARK7) in adipogenesis and inflammation related to obesity in vitro and in vivo. Many intracellular functions of DJ-1, including oxidative stress regulation, are known. However, the possibility of DJ-1 involvement in metabolic disease is largely unknown. Our results suggest that DJ-1 deficiency results in reduced adipogenesis and the down-regulation of pro-inflammatory cytokines in vitro. Furthermore, DJ-1-deficient mice show a low-level inflammatory response in the high-fat diet-induced obesity model. These results indicate previously unknown functions of DJ-1 in metabolism and therefore suggest that precise regulation of DJ-1 in adipose tissue might have a therapeutic advantage for metabolic disease treatment. [ABSTRACT FROM AUTHOR]

We present a single-molecule imaging platform that quantitatively explores the spatiotemporal dynamics of individual insulin receptors in living cells. Modified DNA aptamers that specifically recognize insulin receptors (IRs) with a high affinity were selected through the SELEX process. Using quantum dot-labeled aptamers, we successfully imaged and analyzed the diffusive motions of individual IRs in the plasma membranes of a variety of cell lines (HIR, HEK293, HepG2). We further explored the cholesterol-dependent movement of IRs to address whether cholesterol depletion interferes with IRs and found that cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin reduces the mobility of IRs. The aptamer-based single-molecule imaging of IRs will provide better understanding of insulin signal transduction through the dynamics study of IRs in the plasma membrane. [ABSTRACT FROM AUTHOR]

Biomarker-specific photothermal nanoparticles that can efficiently sense markers that are overexpressed in distinguished adenocarcinomas have attracted much interest in an aspect of efficacy increase of cancer treatment. We demonstrated a promising prospect of a smart photothermal therapy agent employing anti-epidermal growth factor receptor aptamer (AptEGFR)-conjugated polyethylene glycol (PEG) layted gold nanorods (AptEGFR-PGNRs). The cetyltrimethylammonium bromide bilayer on GNRs was replaced with heterobifunctional PEG (COOH-PEG-SH) not only to serve as a biocompatible stabilizer and but also to conjugate AptEGFR. Subsequently, to direct photothermal therapy agent toward epithelial cancer cells, the carboxylated PEGylated GNRs (PGNRs) were further functionalized with AptEGFR using carbodiimide chemistry. Then, to assess the potential as biomarker-specific photothermal therapy agent of synthesized AptEGFR-PGNRs, the optical properties, bio-compatibility, colloidal stability, binding affinity, and epicellial cancer cell killing efficacy in vitro/in vivo under near-infrared laser irradiation were investigated. As a result, AptEGFR-PGNRs exhibit excellent tumor targeting ability and feasibility of effective photothermal ablation cancer therapy. [ABSTRACT FROM AUTHOR]

Physical inactivity can lead to obesity and fat accumulation in various tissues. Critical complications of obesity include type II diabetes and nonalcoholic fatty liver disease (NAFLD). Exercise has been reported to have ameliorating effects on obesity and NAFLD. However, the underlying mechanism is not fully understood. We showed that liver expression of macrophage migration inhibitory factor (MIF) was increased after 4 weeks of treadmill exercise. Phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase in human hepatocyte cell lines was enhanced after MIF treatment. These responses were accompanied by increases in lipid oxidation. Moreover, inhibition of either AMPKor cluster of differentiation 74 resulted in inhibition of MIF-induced lipid oxidation. Furthermore, the administration of MIF to a human hepatocyte cell line and mice liver reduced liver X receptor agonist-induced lipid accumulation. Taken together, these results indicate that MIF is highly expressed in the liver during physical exercise and may prevent hepatic steatosis by activating the AMPK pathway. [ABSTRACT FROM AUTHOR]

Purpose: Acute pancreatitis is one of the potentially lethal complications that occurs after cardiac surgery. We tried to identify risk factors for and the prognosis of acute pancreatitis after cardiac valve surgery with cardiopulmonary bypass. Materials and Methods: We retrospectively analyzed a database of consecutive patients who underwent cardiac valve surgery with cardiopulmonary bypass between January 2005 and April 2010 at our institution. Patients were classified as having acute pancreatitis based on serum lipase concentration and clinical symptoms (lipase ≥180 U/L or ≥60 U/L with relevant symptoms). Results: Of the 986 patients who underwent cardiac valve surgery with cardiopulmonary bypass, 58 (5.9%) patients developed post-operative pancreatitis. Post-operative hospital stay was significantly longer (29.7±45.6 days vs. 12.4±10.7 days, p=0.005) and in-hospital mortality rate was higher (15.5% vs. 2.0%, p<0.001) in patients with post-operative pancreatitis than those without. Hypertension, chronic kidney disease, and peri-operative use of norepinephrine were identified as independent risk factors for developing pancreatitis after cardiac valve surgery. Conclusion: We found that acute pancreatitis after cardiac valve surgery requires longer hospitalization and increases the in-hospital mortality rate. Clinicians should be aware that patients could develop pancreatitis after cardiac valve surgery, especially in patients with hypertension and chronic kidney disease treated with norepinephrine. [ABSTRACT FROM AUTHOR]

Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif-/- mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain. [ABSTRACT FROM AUTHOR]

The mammalian target of rapamycin complex 1 (mTORC1) is a molecular hub that regulates protein synthesis in response to a number of extracellular stimuli. Cyclic AMP (cAMP) is considered to be an important second messenger that controls mTOR; however, the signaling components of this pathway have not yet been elucidated. Here, we identify cAMP phosphodiesterase 4D (PDE4D) as a binding partner of Rheb that acts as a cAMP-specific negative regulator of mTORC1. Under basal conditions, PDE4D binds Rheb in a noncatalytic manner that does not require its cAMP-hydrolyzing activity and thereby inhibits the ability of Rheb to activate mTORC1. However, elevated cAMP levels disrupt the interaction of PDE4D with Rheb and increase the interaction between Rheb and mTOR. This enhanced Rheb-mTOR interaction induces the activation of mTORC1 and cap-dependent translation, a cellular function of mTORC1. Taken together, our results suggest a novel regulatory mechanism for mTORC1 in which the cAMP-determined dynamic interaction between Rheb and PDE4D provides a key, unique regulatory event. We also propose a new role for PDE4 as a molecular transducer for cAMP signaling. [ABSTRACT FROM AUTHOR]

The article discusses a study which investigated the role of neuropilin-I (NP-1), a vascular endothelial growth factor receptor (VEGFR), in rheumatoid inflammation. The study also examined if blockade of NP-1 can suppress synoviocyte survival and angiogenesis. It found that anti-NP-1 peptide suppresses VEGF165>-induced increases in synoviocyte survival and angiogenesis. Study authors concluded that anti-NP-1 peptide could be used in alleviating chronic arthritis.

The article focuses on a study which investigated if sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, regulates synoviocyte hyperplasia and T cell activation in rheumatoid arthritis (RA). Study authors analyzed synoviocyte survival, levels of Bcl-2, Bax, p53, and pAkt, and cytokine concentrations in culture supernatants from mononuclear cells. They found that SFN inhibit synovial hyperplasia, activate T cell proliferation and the production of interleukin (IL)-17 and tumor necrosis factorα.

Chemically modified nucleotides have been developed and applied into SELEX procedure to find a novel type of aptamers to fit with targets of interest. In this study, we directly performed chemical modification of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (called 5-BzdU) in the AS1411 aptamer, which binds to the nucleolin protein expressed in cancer cells. Forty-seven compounds of AS1411-containing Cy3-labeled 5-BzdU (called Cy3-(5-BzdU)-modified-AS1411) were synthesized by randomly substituting thymidines one to twelve in AS1411 with Cy3-labeled 5-BzdU. Both statistically quantified fluorescence measurements and confocal imaging analysis demonstrated at least three potential compounds of interest: number 12, 29 and 41 that significantly increased the targeting affinity to cancer cells but no significant activity from normal healthy cells. These results suggest that the position and number of substituents in AS1411 are critical parameters to improve the aptamer function. In this study, we demonstrated that chemical modification of the existing aptamers enhanced the binding and targeting affinity to targets of interest without additional SELEX procedures. [ABSTRACT FROM AUTHOR]

Background: Upon ligand binding, cell surface signaling receptors are internalized through a process tightly regulated by endocytic proteins and adaptor protein 2 (AP2) to orchestrate them. Although the molecular identities and roles of endocytic proteins are becoming clearer, it is still unclear what determines the receptor endocytosis kinetics which is mainly regulated by the accumulation of endocytic apparatus to the activated receptors. Methodology/Principal Findings: Here we employed the kinetic analysis of endocytosis and adaptor recruitment to show that μ2, a subunit of AP2 interacts directly with phospholipase D (PLD)1, a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). We also demonstrate that the PLD1-m2 interaction requires the binding of PLD1 with phosphatidic acid, its own product. Conclusions/Significance: These results suggest that the temporal regulation of EGFR endocytosis is achieved by autoregulatory PLD1 which senses the receptor activation and triggers the translocation of AP2 near to the activated receptor. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid (LPA) is known to have diverse cellular effects, but although LPA is present in many biological fluids, including blood, its effects on glucose metabolism have not been elucidated. In this study, we investigated whether LPA stimulation is related to glucose regulation. LPA was found to enhance glucose uptake in a dose-dependent manner both in L6 GLUT4 myc myotubes and 3T3-L1 adipocytes by triggering GLUT4 translocation to the plasma membrane. Moreover, the effect of LPA on glucose uptake was completely inhibited by pretreating both cells with LPA receptor antagonist Ki16425 and Gi inhibitor pertussis toxin. In addition, LPA increased the phosphorylation of AKT-1 with no effects on IRS-1, and LPA-induced glucose uptake was abrogated by pretreatment with the PI 3-kinase inhibitor LY294002. When low concentration of insulin and LPA were treated simultaneously, an additive effect on glucose uptake was observed in both cell types. In line with its cellular functions, LPA significantly lowered blood glucose levels in normal mice but did not affect insulin secretion. LPA also had a glucose-lowering effect in streptozotocin-treated type 1 diabetic mice. In combination, these results suggest that LPA is involved in the regulation of glucose homeostasis in muscle and adipose tissues. [ABSTRACT FROM AUTHOR]

Growth hormone binds to its membrane receptor (GHR), whereby it regulates many cellular functions, including proliferation, differentiation and chemotaxis. However, although the activation of growth hormone-mediated signalling is well understood, the precise mechanism responsible for its regulation has not been elucidated. Here, we demonstrate that phospholipase Cγ1 (PLCγ1) modulates the action of growth hormone-mediated signalling by interacting with tyrosine kinase Jak2 (janus kinase 2) in a growth hormone-dependent manner. In the absence of PLCγ1 (PLCγ1−/−), growth hormone-induced JAK2 and STAT5 phosphorylation significantly increased in mouse embryonic fibroblasts (MEFs). Furthermore, the re-expression of PLCγ1 reduced growth hormone-induced Jak2 activation. Growth hormone-induced Jak2 phosphorylation was enhanced by siRNA-specific knockdown of PLCγ1. Interestingly, PLCγ1 physically linked Jak2 and protein tyrosine phosphatase-1B (PTP-1B) by binding to both using different domains, and this process was implicated in the modulation of cytokine signalling through Jak2. In addition, in PLCγ1−/− MEFs, growth hormone-dependent c-Fos activation was upregulated and growth hormone-induced proliferation was potentiated. These results suggest that PLCγ1 has a key function in the regulation of growth hormone-mediated signalling by negatively regulating Jak2 activation. [ABSTRACT FROM AUTHOR]

WAVE1—the Wiskott–Aldrich syndrome protein (WASP)-family verprolin homologous protein 1—is a key regulator of actin-dependent morphological processes in mammals, through its ability to activate the actin-related protein (Arp2/3) complex. Here we show that WAVE1 is phosphorylated at multiple sites by cyclin-dependent kinase 5 (Cdk5) both in vitro and in intact mouse neurons. Phosphorylation of WAVE1 by Cdk5 inhibits its ability to regulate Arp2/3 complex-dependent actin polymerization. Loss of WAVE1 function in vivo or in cultured neurons results in a decrease in mature dendritic spines. Expression of a dephosphorylation-mimic mutant of WAVE1 reverses this loss of WAVE1 function in spine morphology, but expression of a phosphorylation-mimic mutant does not. Cyclic AMP (cAMP) signalling reduces phosphorylation of the Cdk5 sites in WAVE1, and increases spine density in a WAVE1-dependent manner. Our data suggest that phosphorylation/dephosphorylation of WAVE1 in neurons has an important role in the formation of the filamentous actin cytoskeleton, and thus in the regulation of dendritic spine morphology. [ABSTRACT FROM AUTHOR]

Here we report inhibition of phospholipase C-β1 (PLC-β1)-mediated signaling by post-translational glycosylation with β-N-acetylglucosamine (O-GlcNAc modification). In C2C12 myoblasts, isoform-specific knock-down experiments using siRNA showed that activation of bradykinin (BK) receptor led to stimulation of PLC-β1 and subsequent intracellular Ca2+ mobilization. In C2C12 myotubes, O-GlcNAc modification of PLC-β1 was markedly enhanced in response to treatment with glucosamine (GlcNH2), an inhibitor of O-GlcNAase (PUGNAc) and hyperglycemia. This was associated with more than 50% inhibition of intracellular production of IP3 and Ca2+ mobilization in response to BK. Since the abundance of PLC-β1 remained unchanged, these data suggest that O-GlcNAc modification of PLC-β1 led to inhibition of its activity. Moreover, glucose uptake stimulated by BK was significantly blunted by treatment with PUGNAc. These data support the notion that O-GlcNAc modification negatively modulates the activity of PLC-β1. J. Cell. Physiol. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST–Src–SH2 fusion proteins. We found MVP as a Src–SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades. [ABSTRACT FROM AUTHOR]

Endogenous opioid peptides, found in the central and peripheral nervous systems, perform neuromodulatory roles, and display a wide range of functional and pharmacological properties in vitro and in vivo. In this study, we investigated the effects of prodynorphin gene products on intracellular signaling events and cell survival in rat pheochromocytoma PC12 cells. Leumorphin, but not other prodynorphin gene products including dynorphin A, β-neoendorphin and rimorphin (dynorphin B), increased cell viability in PC12 cells. The cytoprotective effect of leumorphin was dependent on the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, but was insensitive to both naloxone, a general antagonist of the opioid receptor, and nor-binaltorphimine, a specific antagonist of the kappa opioid receptor. Moreover, a competition-binding assay clearly revealed that leumorphin had another binding site(s) in addition to that for the kappa opioid receptor. Interestingly, leumorphin induced activation of the epidermal growth factor receptor via a Src-dependent mechanism, which was proved to be responsible for the increased survival response. Flow cytometric and microscopic analysis showed that leumorphin rescued cells from serum deprivation-induced apoptosis. Collectively, we suggest that leumorphin prevents apoptosis via epidermal growth factor receptor-mediated activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, which occur independent of the kappa opioid receptor. [ABSTRACT FROM AUTHOR]

Summarizes the results of a study on the properties of the phox homology (PX) domains of Phospholipase D (PLD) 1 and PLD2 in terms of phosphoinositide binding and PLD activity regulation. Effect of the mutation of the conserved arginine at position 179 of the PLD1 PX domain to lysine or to alanine on Phosphatidylinositol (3,4,5)-triphosphate (P3) binding; Factor that stimulates the enzymatic activity of PLD1; Ability of PLD1 PX domain to enable PLD1 to mediate signal transduction via extracellular signal-regulated kinases 1/2 by providing a direct binding site for Phosphatidylinositol (3,4,5)P3 and by activating PLD1.

It has been established that protein kinase Cζ (PKCζ) participates in diverse signaling pathways and cellular functions in a wide variety of cells, exhibiting properties relevant to cellular survival and proliferation. Currently, however, the regulation mechanism of PKCζ remains elusive. Here, for the first time, we determine that phospholipase D2 (PLD2) enhances PKCζ activity through direct interaction in a lipase activity-independent manner. This interaction of the PLD2-Phox homology (PX) domain with the PKCζ-kinase domain also induces the activation loop phosphorylation of PKCζ and downstream signal stimulation, as measured by pT0 86 kinase phospborylation. Furthermore, only the PLD2-PX domain directly stimulates PKCζ activity in vitro, and it is necessary for the formation of the ternary complex with phosphoinositide-dependent kinase 1 and PKCζ. The mutant that substitutes the triple lysine residues (Lys101, Lysl02, and Lys103) within the PLD2-PX domain with alanine abolishes interaction with the PKCζ-kinase domain and activation of PKCζ. Moreover, breast cancer cell viability is significantly affected by PLD2 silencing. Taken together, these results suggest that the PLD2-mediated PKCζ activation is induced by its PX domain performing both direct activation of PKCζ and assistance of activation loop phosphorylation. Furthermore, we find it is an important factor in the survival of breast cancer cells. [ABSTRACT FROM AUTHOR]

In contrast to the 1980s, we find substantial increases in the homeownership rates of young adults in the 1990s. Focusing on the younger half of the baby boom generation, aged 35 to 44 in 2000, we explore the factors that caused steeper trajectories into homeownership in some metropolitan areas. Factors include prices and incomes, housing construction relative to employment growth, and rates of household formation and immigration. Homeownership gains are modeled separately for whites, blacks, Asians, and Hispanics. Our findings highlight the importance of household formation on regional homeownership rates. Evidence shows greater homeownership gains in areas with greater rent increases, indicating lower relative costs of owning, and with greater price increases, indicating greater investment incentives. Our findings also underscore the importance of keeping housing construction consistent with employment growth. Finally, the effect of immigration was especially important for Hispanics, sharply depressing homeownership in regions with more recently arrived immigrants. [ABSTRACT FROM AUTHOR]