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mTORC1 is a protein kinase important for metabolism and is regulated by growth factor and nutrient signaling pathways, mediated by the Rheb and Rag GTPases, respectively. Here we provide the first animal model in which both pathways were upregulated through concurrent mutations in their GTPase-activating proteins, Tsc1 and Depdc5 . Unlike former models that induced limited mTORC1 upregulation, hepatic deletion of both Tsc1 and Depdc5 (DKO) produced strong, synergistic activation of the mTORC1 pathway and provoked pronounced and widespread hepatocyte damage, leading to externally visible liver failure phenotypes, such as jaundice and systemic growth defects. The transcriptome profile of DKO was different from single knockout mutants but similar to those of diseased human livers with severe hepatitis and mouse livers challenged with oxidative stress-inducing chemicals. In addition, DKO liver cells exhibited prominent molecular pathologies associated with excessive endoplasmic reticulum (ER) stress, oxidative stress, DNA damage and inflammation. Although DKO liver pathologies were ameliorated by mTORC1 inhibition, ER stress suppression unexpectedly aggravated them, suggesting that ER stress signaling is not the major conduit of how hyperactive mTORC1 produces liver damage. Interestingly, superoxide scavengers N-acetylcysteine (NAC) and Tempol, chemicals that reduce oxidative stress, were able to recover liver phenotypes, indicating that mTORC1 hyperactivation induced liver damage mainly through oxidative stress pathways. Our study provides a new model of unregulated mTORC1 activation through concomitant upregulation of growth factor and nutrient signaling axes and shows that mTORC1 hyperactivation alone can provoke oxidative tissue injury., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2019.)

N 6 -methyladenosine (m 6 A) is the most prevalent modified nucleotide in mRNA 1,2 , with around 25% of mRNAs containing at least one m 6 A. Methylation of mRNA to form m 6 A is required for diverse cellular and physiological processes 3 . Although the presence of m 6 A in an mRNA can affect its fate in different ways, it is unclear how m 6 A directs this process and why the effects of m 6 A can vary in different cellular contexts. Here we show that the cytosolic m 6 A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m 6 A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m 6 A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m 6 A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m 6 A-modified mRNAs are governed by liquid-liquid phase separation principles.

Cheonggukjang and chaga mushrooms have numerous health benefits, and have been used in alternative medicine. Therefore, a powder mixture of 98: Cheonggukjang and 2: Chaga extracts was fermented with Lactobacillus acidophilus KCTC3925 (FCC) and its anti-obesity effects in high-fat diet (HFD)-induced obese mice were determined. Five-week-old male ICR mice were fed a normal diet or HFD in the presence or absence of 3% and 5% FCC by weight ( n = 10 per group). After 12 weeks, the mice were sacrificed, and the serum and tissue samples were collected for analysis. Body weight and epididymal fat pad weight were significantly lowered in the 3% and 5% FCC groups compared with those in the HFD control group ( p < 0.01). FCC supplementation suppressed serum triglyceride and increased serum HDL-C levels ( p < 0.01). Serum GOT, GPT, and leptin levels, hepatic COX-2 mRNA expression, and splenic COX-2 and IL-4 mRNA expression were significantly higher in the HFD groups than in the control group ( p > 0.05); however, except for splenic IL-4 levels, the increases were significantly attenuated by FCC supplementation. Expression of ICAM-1, an aortic inflammatory marker, was significantly increased in the HFD group; this effect was suppressed in the 3% FCC group ( p < 0.01) but not in the 5% FCC group. FCC suppressed the body weight and epididymal fat pad weight gain, as well as inflammatory responses in the liver and spleen of HFD-fed mice. Thus, FCC supplementation will be beneficial for the treatment of obesity-related effects.

Macroautophagy (hereafter autophagy) is a well-conserved cellular process through which cytoplasmic components are delivered to the vacuole/lysosome for degradation and recycling. Studies have revealed the molecular mechanism of transcriptional regulation of autophagy-related (ATG) genes upon nutrient deprivation. However, little is known about their translational regulation. Here, we found that Dhh1, a DExD/H-box RNA helicase, is required for efficient translation of Atg1 and Atg13, two proteins essential for autophagy induction. Dhh1 directly associates with ATG1 and ATG13 mRNAs under nitrogen-starvation conditions. The structured regions shortly after the start codons of the two ATG mRNAs are necessary for their translational regulation by Dhh1. Both the RNA-binding ability and helicase activity of Dhh1 are indispensable to promote Atg1 translation and autophagy. Moreover, eukaryotic translation initiation factor 4E (EIF4E)-associated protein 1 (Eap1), a target of rapamycin (TOR)-regulated EIF4E binding protein, physically interacts with Dhh1 after nitrogen starvation and facilitates the translation of Atg1 and Atg13. These results suggest a model for how some ATG genes bypass the general translational suppression that occurs during nitrogen starvation to maintain a proper level of autophagy., Competing Interests: The authors have declared that no competing interests exist.

Post-transcriptional RNA processing is a core mechanism of gene expression control in cell stress response. The poly(A) tail influences mRNA translation and stability, but it is unclear whether there are global roles of poly(A)-tail lengths in cell stress. To address this, we developed tail-end displacement sequencing (TED-seq) for an efficient transcriptome-wide profiling of poly(A) lengths and applied it to endoplasmic reticulum (ER) stress in human cells. ER stress induced increases in the poly(A) lengths of certain mRNAs, including known ER stress regulators, XBP1, DDIT3, and HSPA5. Importantly, the mRNAs with increased poly(A) lengths are both translationally de-repressed and stabilized. Furthermore, mRNAs in stress-induced RNA granules have shorter poly(A) tails than in the cytoplasm, supporting the view that RNA processing is compartmentalized. In conclusion, TED-seq reveals that poly(A) length is dynamically regulated upon ER stress, with potential consequences for both translation and mRNA turnover., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

AMP-activated protein kinase (AMPK) regulates autophagy initiation when intracellular ATP level decreases. However, the role of AMPK during autophagosome maturation is not fully understood. Here, we report that AMPK contributes to efficient autophagosome maturation and lysosomal fusion. Using CRISPR-Cas9 gene editing, we generated AMPK α1 knockout HEK293T cell lines, in which starvation-induced autophagy is impaired. Compound C, an AMPK-independent autophagy inducer, and trehalose, an mTOR-independent autophagy inducer were used to examine the role of AMPK in autophagosome maturation and lysosomal fusion. While the treatment of control cells with either compound C or trehalose induces activation of autophagosomes as well as autolysosomes, the treatment of AMPK α1 knockout cells with compound C or trehalose induces mainly activation of autophagosomes, but not autolysosomes. We demonstrate that this effect is due to interference with the fusion of autophagosomes with lysosomes in AMPK α1 knockout cells. The transient expression of AMPK α1 can rescue autophagosome maturation. These results indicate that AMPK α1 is required for efficient autophagosome maturation and lysosomal fusion.

The anti-melanogenic effects of the extract of Angelica tenuissima (AT) root and the extract of AT root fermented by Aspergillus oryzae (FAT) were investigated. These effects were determined by measuring the inhibitory activity of AT and FAT on melanin production in B16F10 melanocytes and with in vitro tyrosinase activity assays. The AT extract inhibited melanin production at concentrations above 250 μg/ml, and this inhibitory effect was significantly enhanced by the fermentation process with A. oryzae . HPLC analysis resulted in the isolation of two active compounds from both the AT and FAT extracts. Their chemical structures were identified as decursin and Z-ligustilide through comparison with previously reported NMR data. The decursin and Z-ligustilide contents were increased in the FAT extract and could be responsible for its enhanced inhibitory effects on melanin production and tyrosinase activity compared with that of the AT extract.

Upon stress, cytoplasmic mRNA is sequestered to insoluble ribonucleoprotein (RNP) granules, such as the stress granule (SG). Partially due to the belief that translationally suppressed mRNAs are recruited to SGs in bulk, stress-induced dynamic redistribution of mRNA has not been thoroughly characterized. Here, we report that endoplasmic reticulum (ER) stress targets only a small subset of translationally suppressed mRNAs into the insoluble RNP granule fraction (RG). This subset, characterized by extended length and adenylate-uridylate (AU)-rich motifs, is highly enriched with genes critical for cell survival and proliferation. This pattern of RG targeting was conserved for two other stress types, heat shock and arsenite toxicity, which induce distinct responses in the total cytoplasmic transcriptome. Nevertheless, stress-specific RG-targeting motifs, such as guanylate-cytidylate (GC)-rich motifs in heat shock, were also identified. Previously underappreciated, transcriptome profiling in the RG may contribute to understanding human diseases associated with RNP dysfunction, such as cancer and neurodegeneration., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Autophagy is one of the major degradative mechanisms that can eliminate excessive nutrients, toxic protein aggregates, damaged organelles and invading microorganisms. In response to obesity and obesity-associated lipotoxic, proteotoxic and oxidative stresses, autophagy plays an essential role in maintaining physiological homeostasis. However, obesity and its associated stress insults can often interfere with the autophagic process through various mechanisms, which result in further aggravation of obesity-related metabolic pathologies in multiple metabolic organs. Paradoxically, inhibition of autophagy, within specific contexts, indirectly produces beneficial effects that can alleviate several detrimental consequences of obesity. In this minireview, we will provide a brief discussion about our current understanding of the impact of obesity on autophagy and the role of autophagy dysregulation in modulating obesity-associated pathological outcomes.

Crosstalk between adipocytes and macrophages has been suggested to play a crucial role in metabolic disorders such as obesity, insulin resistance, and type 2 diabetes. The objective of this study was to evaluate the effect of nobiletin on the interaction between adipocytes and macrophages. The results showed that nobiletin significantly and dose-dependently inhibited the secretion of inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor (TNF-α), and monocyte chemoattractant protein (MCP)-1, in a coculture of adipocytes and macrophages. The expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), in differentiated 3T3-L1 cells cocultured in transwell system was blocked by nobiletin. Nobiletin also downregulated the expression of inducible NO synthase in cocultured differentiated RAW264.7 cells. Furthermore, heme oxygenase-1 (HO-1) was significantly induced by nobiletin treatment in both cell types, and small interfering (si) RNA-mediated knockdown of HO-1 significantly recovered the inhibitory effects of nobiletin on the NO production in cocultured cells. These results suggest that nobiletin exerts anti-inflammatory effects on the crosstalk between adipocytes and macrophages by inducing HO-1. Nobiletin may have potential for the prevention of obesity-related metabolic diseases.

[Purpose] The purpose of this study is to analyze changes in movements of the neck, trunk and hip according to foot position while performing sit-to-stand (STS) exercises from a height-fixed chair. [Subjects and Methods] The study subjects consisted of 22 university students (12 males and 10 females). STS was conducted using a height-fixed chair at three positions: symmetric foot position, right foot position, and left foot position. Through three-dimensional motion analyzer, the movements of the neck, trunk, and hip were analyzed. [Results] While performing STS, the height was more influential on changed in angle of the neck, trunk, and hip. Moreover, when the height of the chair and the height of the subject were not matched correctly, more effective STS could be achieved when both of feet were laid symmetrically rather than at the other two positions. [Conclusion] It is necessary to employ an appropriate chair height that is matched with the height of the patients when therapy using STS is performed.

Excessive accumulation of reactive oxygen species (ROS) and chronic activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) are well-characterized promoters of aging and age-associated degenerative pathologies. Sestrins, a family of highly conserved stress-inducible proteins, are important negative regulators of both ROS and mTORC1 signaling pathways; however, the mechanistic basis of how Sestrins suppress these pathways remains elusive. In the past couple of years, breakthrough discoveries about Sestrin signaling and its molecular nature have markedly increased our biochemical understanding of Sestrin function. These discoveries have also uncovered new potential therapeutic strategies that may eventually enable us to attenuate aging and age-associated diseases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagic adapter, accumulated in the autophagosomes. Reactive oxygen species such as hydrogen peroxide (H2O2) result in muscle cell death by inducing excessive autophagic flux. Treatment with conessine inhibited H2O2-induced autophagic flux in C2C12 myoblast cells and also interfered with cell death. Our results indicate that conessine has the potential effect to inhibit muscle cell death by interfering with autophagic flux.

The mTOR complex 1 (mTORC1) and endoplasmic reticulum (ER) stress pathways are critical regulators of intestinal inflammation and colon cancer growth. Sestrins are stress-inducible proteins, which suppress both mTORC1 and ER stress; however, the role of Sestrins in colon physiology and tumorigenesis has been elusive due to the lack of studies in human tissues or in appropriate animal models. In this study, we show that human SESN2 expression is elevated in the colon of ulcerative colitis patients but is lost upon p53 inactivation during colon carcinogenesis. In mouse colon, Sestrin2 was critical for limiting ER stress and promoting the recovery of epithelial cells after inflammatory injury. During colitis-promoted tumorigenesis, Sestrin2 was shown to be an important mediator of p53's control over mTORC1 signaling and tumor cell growth. These results highlight Sestrin2 as a novel tumor suppressor, whose downregulation can accelerate both colitis and colon carcinogenesis.

[Purpose] The purpose of this study was to determine the difference in isokinetic muscle function in football athletes with a knee injury with and without kinesio taping. [Subjects] The subjects for this study were 10 football athletes (males) with a knee injury. [Methods] Measurements were performed by using Cybex dynamometer under uniform motion before and after the application of kinesio tape to the quadriceps and hamstring muscle. Maximal concentric knee extension and flexion at three angular velocities (60°/s, 120°/s, and 180°/s) were measured. [Results] A significant difference was found in peak torque and total work of the flexion at 120°/s and 180°/s, as well as in the average power of extension at 180°/s. [Conclusion] Though it is not the main therapy for muscle function in football athletes with injury, kinesio taping was an effective adjunct therapy.

[Purpose] The purpose of this study was to ergonomically evaluate the work posture of dentists to examine their subsequent risk of developing musculoskeletal diseases. [Subjects and Methods] Scenes in which the three dentists performed procedures at their dental clinics were videotaped. The videotapes of the dentists' work postures were evaluated and analyzed by using the Rapid Upper Limb Assessment (RULA) and Quick Exposure Check (QEC). [Results] The RULA analysis of the dentists' work posture indicated, "improvement required" in the posture used to treat the anterior and "instant improvement required" in the posture used to treat the maxillary second molar. Of all the work postures studied, the risk was considered particularly high in the lower back and neck, implying prominent problems in these body parts. The QEC analysis showed that the worst work posture was that required to treat the maxillary second molar, which led to a high risk of neck problems and vibrations. [Conclusion] The neck area has the highest risk of developing musculoskeletal disease. Hence, regular rests and the provision of information regarding muscle strengthening exercise for the neck are necessary.

[Purpose] The purpose of this study was to identify the effect of different sitting positions on the skin temperature of the lower extremity. [Subjects] The subjects of this study were 23 healthy university students (8 males, 15 females). [Methods] Normal sitting (NS), upper leg cross (ULC) and ankle on knee (AOK) positions were conducted to measure the changes in skin temperature using digital infrared thermographic imaging (DITI). [Results] ULC upper ankle, NS upper shin, ULC upper shin and NS lower shin showed significant declines in temperature with time. [Conclusion] These finding suggest that the ULC and NS sitting positions cause decline of blood flow volume to the lower extremity resulting in decrease of temperature of the lower extremity. Especially, sitting with the legs crossed interferes with the circulation of blood flowing volume much more than just sitting in a chair.

Reserpine is a well-known medicine for the treatment of hypertension and schizophrenia, but its administration can induce Parkinson's disease (PD)-like symptoms in humans and animals. Reserpine inhibits the vesicular transporter of monoamines and depletes the brain of monoamines such as dopamine. However, the cellular function of reserpine is not fully understood. In this report, we present one possible mechanism by which reserpine may contribute to PD-like symptoms. Reserpine treatment induced the formation of enlarged autophagosomes by inhibiting the autophagic flux and led to accumulation of p62, an autophagy adapter molecule. In particular, reserpine treatment increased the level of α-synuclein protein and led to accumulation of α-synuclein in autophagosomes. Treatment with rapamycin enhanced the effect of reserpine by further increasing the level of α-synuclein and neuronal cell death. Drosophila raised on media containing reserpine showed loss of dopaminergic neurons. Furthermore, cotreatment with reserpine and rapamycin aggravated the loss of dopaminergic neurons. Our results suggest that reserpine contributes to the loss of dopaminergic neurons by interfering with autophagic flux., (Copyright © 2015 Elsevier Inc. All rights reserved.)

[Purpose] The purpose of this study was to analyze the effect of superficial trunk muscle exercise and deep trunk muscle exercise on the foot pressure of healthy adults. [Subjects] The subjects were 30 healthy females and males who agreed to participate in this study. There were two groups, a superficial trunk muscle exercise group and a deep trunk muscle exercise group, with 15 participants in each. [Methods] The exercises were conducted 5 times a week for 4 weeks for both groups. A gait analyzer was used to measure foot plantar pressure while walking on a plate. Participants were measured before starting the exercise and after 4 weeks. The paired t-test was used to analyze the pre-and post-test results. [Results] There were no significant differences in foot pressure in any region in the superficial trunk muscle exercise group. In the deep trunk muscle exercise group, there were statistically significant increase in F1, F4, F5, R1 and R3. In addition, there were significant decreases in R2 and R4. [Conclusion] After the 4-week deep trunk muscle exercise group decreases in foot pressure on the inner foot and increases on the outside of the feet indicate normal and overall even distribution of body weight on the feet.

Fucoidan is an l-fucose-enriched sulfated polysaccharide isolated from brown algae and marine invertebrates. In this study, we investigated the protective effect of fucoidan from Fucus vesiculosus on alcohol-induced murine liver damage. Liver injury was induced by oral administration of 25% alcohol with or without fucoidan (30 mg/kg or 60 mg/kg) for seven days. Alcohol administration increased serum aspartate aminotransferase and alanine aminotransferase levels, but these increases were suppressed by the treatment of fucoidan. Transforming growth factor beta 1 (TGF-β1), a liver fibrosis-inducing factor, was highly expressed in the alcohol-fed group and human hepatoma HepG2 cell; however, the increase in TGF-β1 expression was reduced following fucoidan administration. Treatment with fucoidan was also found to significantly reduce the production of inflammation-promoting cyclooygenase-2 and nitric oxide, while markedly increasing the expression of the hepatoprotective enzyme, hemeoxygenase-1, on murine liver and HepG2 cells. Taken together, the antifibrotic and anti-inflammatory effects of fucoidan on alcohol-induced liver damage may provide valuable insights into developing new therapeutics or interventions.

The fluorescent-antibody-to-membrane-antigen (FAMA) test is regarded as the "gold standard" to detect protective antibodies to varicella-zoster virus (VZV) because of its high sensitivity and specificity. Because the classic FAMA test uses an infectious virus for detection of antibodies to VZV, it is labor-intensive, and also requires special equipment for handling the virus. For this reason, we attempted to develop a simple and safe FAMA assay. Because VZV glycoprotein E (gE) is one of the major VZV glycoproteins, we used the gE protein for the FAMA test (gE FAMA). Here, we demonstrate that overexpression of gE in HEK293T cells can be used to measure antibodies in human serum, and that gE FAMA titers are closely correlated with gpEIA ELISA data. These results indicate that our gE FAMA test has the potential to measure antibodies to VZV.

The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

Fruits of the Litsea family of trees and shrubs contain biologically active compounds, some of which have been used as natural nutrients and flavoring agents in food. In this study, we identified novel anti-nociceptive effects of the 30% ethanol extract, the CH(2)Cl(2) fraction and the associated active components (Hamabiwalactone A and B) from Litsea japonica fruit by using in vivo peripheral and central nervous pain models. In addition, we compared the anti-inflammatory effects of several fractions from L. japonica fruit extracts using lipopolysaccharide (LPS)-stimulated Raw264.7 cells. The CH(2)Cl(2) fraction of L. japonica fruit (LJM) had an optimal combination of anti-inflammatory effects and low cytotoxicity. Dose response studies were performed to determine the inhibitory effects of LJM on the pro-inflammatory enzymes, COX-2/PGE(2) and NO/iNOS, and pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α. Molecular profiling revealed that LJM exerts anti-inflammatory effects through inhibition of NF-κB and JNK/p38 MAPK signaling in LPS-induced macrophages. This study suggests that CH2Cl2 fraction of L. japonica fruit and its bioactive components are potential candidates as anti-inflammatory and analgesic agents (painkillers) for the treatment of inflammatory diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Obesity is associated with chronic low-grade inflammation of adipose tissue. In this study, we investigated the anti-inflammatory effects of esculetin (ECT) through up-regulation of heme oxygenase-1 (HO-1) in cocultured macrophages and adipocytes. RAW264.7 macrophages and differentiated 3T3-L1 adipocytes were cocultured in serum-free Dulbecco's modified Eagle's medium with or without ECT for 24 h. Nitric oxide (NO), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) production was measured in the coculture supernatant. ECT decreased the secretion of NO, TNF-α, and MCP-1. The expression of adipogenic proteins, including peroxisome proliferator-activated receptors γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) in cocultured adipocytes and inducible nitric oxide synthase (iNOS) in cocultured macrophages, was inhibited by ECT. Additionally, HO-1 expression was induced in cocultured macrophages and adipocytes. Silencing of HO-1 expression increased the production of NO, TNF-α, and MCP-1 in cocultured cells, in spite of the presence of ECT. This study demonstrated that ECT exhibited anti-inflammatory properties by inhibiting the production of proinflammatory cytokines in the interaction between adipocytes and macrophages through HO-1 expression. ECT may have the potential to improve chronic inflammation in obesity.

Exposure to microgravity is supposed to affect almost all biological systems, and we speculated that microgravity is potentially involved in autophagy regulation. A clinostat was used to simulate microgravity, and HEK293 cells that stably express GFP-LC3 were used for sensitive monitoring of autophagy induction. The clinorotation of GFP-LC3 cells resulted in autophagosome formation in the cytoplasm and a change in autophagosomal marker expression. Autophagy induction was accompanied by phosphorylation of AMPK (Thr 172) and by the dephosphorylation of mammalian target of rapamycin. To elucidate the role of AMPK in microgravity-induced autophagy, we suppressed AMPK expression by knockdown via siRNA, which inhibited the induction of autophagy upon exposure to microgravity. In addition, the clinorotation of C2C12 myotube cells resulted in the enlarged and distinctive LC3 spots in the cytoplasm and AMPK activation. These results indicate that simulated microgravity possibly contributes to autophagy induction by regulating AMPK.

Ethnopharmacological Relevance: Postmenopausal osteoporosis is one of the most common disorders in women after menopause, which is linked to an estrogen deficiency and characterized by an excessive loss of trabecular bone. Rubus coreanus and Astragalus membranaceus have been used for their various pharmacological properties in Asia as a traditional medicine. The present study evaluated the anti-osteoporotic effects of the optimal combination of Rubus coreanus and Astragalus membranaceus in 7:3 mixture (RAM) in ovariectomized (OVX) mice by investigating bone biomechanical properties and the serum levels of TNF-α, osteocalcin, RANKL, OPG, and RANK-RANKL signal-related osteoclast differentiation markers., Materials and Methods: A total of 36 mature female outbred ICR (Institute of cancer research) strain mice (7 weeks) were divided into 6 groups with 7 mice in each group as follows: (1) Sham-operated control mice (Sham) received daily oral phosphate-buffered-saline (PBS) of equal volumes through gavage. (2) OVX mice received a daily oral gavage of PBS (OVX). (3) OVX mice were treated daily with 50mg/kgb.w./day of RAM (4) with 100mg/kgb.w./day of RAM or (5) with 200mg/kgb.w./day of RAM via oral gavage. (6) OVX mice received i.p. injections of 17β-estradiol (E2) (0.1mg/kgb.w./day) three times per week for 12 weeks., Results: Micro-CT images showed that oral administration of RAM to OVX mice prevented tibial bone loss, preserved trabecular bone microarchitecture, and improved bone biomechanical properties. RAM administration also showed recovery effects on the levels of TNF-α, OPG and RANKL concentration in OVX-states. Additionally, we found that the mechanism by which RAM elicited anti-osteoporotic effects was by down-regulating the expression of TRAF6 and NFATc1 in RANKL-RANK pathway, a route of osteoclast differentiation, followed by reducing the production of osteoclast differentiation factors, calcitonin receptors and cathepsin K., Conclusions: Our research strongly suggests that RAM can be clinically used in the prevention and treatment of postmenopausal osteoporosis., (© 2013 Published by Elsevier Ireland Ltd.)

A series of novel di-α-aminophosphonate derivatives were synthesized by a one-pot method in the presence of PEG-H2 O under ultrasonic irradiation and were characterized by IR, (1) H NMR, (13) C NMR, mass spectrometry, and elemental analysis. The newly synthesized compounds were evaluated for their cytotoxic activities against the human lung cancer cell line H1299 and the human breast cancer cell line MCF7 in vitro by the MTT method. All compounds showed moderate cytotoxic activity on both cell lines, and compounds 4b and 4c showed marked activity., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Exposure to gamma radiation causes a wide range of biological damage and alterations, including oxidative stress, inflammation and cancer. This study aimed to identify the radioprotective effect of Korean red ginseng extract (RG) against whole-body gamma-irradiation (γIR) in mice and the regulatory mechanisms of the radiosensitive gene in spleen, cyclooxygenase-2 (COX-2). RG was administered intraperitoneally (i.p.) or orally (p.o.) to C57BL/6 mice for five days, which were then exposed to 6.5 Gy of (137)Cs-γIR. Thymus and spleen were harvested after three days, and organ size and COX-2 expression of the spleen using Western blotting, were examined. γIR shrank both organs and RG recovered the size of thymus but not spleen. RG also significantly inhibited the increased expression of COX-2 induced by γIR. These results were similar following both routes of RG administration, however i.p. RG administration was more effective, thus it was used in progressive studies. In terms of COX-2 expression related intracellular factors, we found here that γIR activated the p38 MAPK, PI3K/Akt and HO-1 but not NF-κB or Nrf2. Activated p38 MAPK, PI3K/Akt and HO-1 were down-regulated by RG while the RG-induced COX-2 expression was only related to HO-1 activation. These results suggest that RG supplementation provides protective effects against radiation-induced inflammation and cancer, and its potential to be utilized in clinical trials and functional foods., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Coriolus versicolor has been known to be an immune stimulator effects. For further understanding of the phagocytic activity and the intracellular mechanisms of β-glucan from C. versicolor (CVG), we examined the phagocytic activity, phosphorylation of Akt and CK2, nucleus translocation of p65 and Ikaros activity in β-glucan-treated macrophages using RT-PCR, western blotting, and IP assay. The role of Ikaros in regulating phagocytic effects of CVG was also determined using Ikaros dominant negative isoform cells. This study suggests that CK2/Ikaros are positive regulators and novel signaling pathway involved in phagocytosis and contributes to elucidating the mechanism underlying phagocytic activity induced by β-glucan., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Nowadays, although its clinical value remains controversial institutions utilize hair mineral analysis. Arguments about the reliability of hair mineral analysis persist, and there have been evaluations of commercial laboratories performing hair mineral analysis., Objective: The objective of this study was to assess the reliability of intra-laboratory and inter-laboratory data at three commercial laboratories conducting hair mineral analysis, compared to serum mineral analysis., Methods: Two divided hair samples taken from near the scalp were submitted for analysis at the same time, to all laboratories, from one healthy volunteer. Each laboratory sent a report consisting of quantitative results and their interpretation of health implications. Differences among intra-laboratory and interlaboratory data were analyzed using SPSS version 12.0 (SPSS Inc., USA)., Results: All the laboratories used identical methods for quantitative analysis, and they generated consistent numerical results according to Friedman analysis of variance. However, the normal reference ranges of each laboratory varied. As such, each laboratory interpreted the patient's health differently. On intra-laboratory data, Wilcoxon analysis suggested they generated relatively coherent data, but laboratory B could not in one element, so its reliability was doubtful. In comparison with the blood test, laboratory C generated identical results, but not laboratory A and B., Conclusion: Hair mineral analysis has its limitations, considering the reliability of inter and intra laboratory analysis comparing with blood analysis. As such, clinicians should be cautious when applying hair mineral analysis as an ancillary tool. Each laboratory included in this study requires continuous refinement from now on for inducing standardized normal reference levels.

Replication protein A (RPA) is a eukaryotic single-stranded DNA binding protein that is essential for DNA replication, repair, and recombination, and human RPA interacting protein α (hRIPα) is the nuclear transporter of RPA. Here, we report the regulatory role of hRIPα protein in cell proliferation. Western blot analysis revealed that the level of hRIPα was frequently elevated in cervical tumors tissues and hRIPα knockdown by siRNA inhibited cellular proliferation through deregulation of the cell cycle. In addition, overexpression of hRIPα resulted in increased clonogenicity. These results indicate that hRIPα is involved in cell proliferation through regulation of RPA transport., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Objectives: The angiogenic drive in skeletal muscle ischemia remains poorly understood. Innate inflammatory pathways are activated during tissue injury and repair, suggesting that this highly conserved pathway may be involved in ischemia-induced angiogenesis. We hypothesize that one of the endogenous ligands for innate immune signaling, high mobility group box 1 (HMGB1), in combination with autophagic responses to hypoxia or nutrient deprivation, plays an important role in angiogenesis., Methods: Human dermal microvascular endothelial cells (ECs) were cultured in normoxia or hypoxia (1% oxygen). Immunocytochemical analysis of HMGB1 subcellular localization, evaluation of tube formation, and Western blot analysis of myotubule light-chain 3I (LC3I) conversion to LC3II, as a marker of autophagy, were conducted. 3-Methyladenine (3MA), chloroquine, or rapamycin were administered to inhibit or promote autophagy, respectively. In vivo, a murine hind limb ischemia model was performed. Muscle samples were collected at 4 hours to evaluate for nuclear HMGB1 and at 14 days to examine endothelial density. Perfusion recovery in the hind limbs was calculated by laser Doppler perfusion imaging (LDPI)., Results: Hypoxic ECs exhibited reduced nuclear HMGB1 staining compared with normoxic cells (mean fluorescence intensity, 186.9 ± 17.1 vs 236.0 ± 1.6, P = .01) with a concomitant increase in cytosolic staining. HMGB1 treatment of ECs enhanced tube formation, an angiogenic phenotype of ECs. Neutralization of endogenous HMGB1 markedly impaired tube formation and inhibited LC3II formation. Inhibition of autophagy with 3MA or chloroquine abrogated tube formation, whereas its induction with rapamycin enhanced tubing and promoted HMGB1 translocation. In vivo, ischemic skeletal muscle showed reduced numbers of HMGB1-positive myocyte nuclei compared with nonischemic muscle (34.9% ± 1.9% vs 51.7% ± 2.0%, P < .001). Injection of HMGB1 into ischemic hind limbs increased perfusion recovery by 21% and increased EC density (49.2 ± 4.1 vs 34.2 ± 3.4 ECs/high-powered field, respectively; P = .02) at 14 days compared with control hind limbs., Conclusions: Nuclear release of HMGB1 and autophagy occur in ECs in response to hypoxia or serum depletion. HMGB1 and autophagy are necessary and likely play an interdependent role in promoting the angiogenic behavior of ECs. In vivo, HMGB1 promotes perfusion recovery and increased EC density after ischemic injury. These findings suggest a possible mechanistic link between autophagy and HMGB1 in EC angiogenic behavior and support the importance of innate immune pathways in angiogenesis., (Published by Mosby, Inc.)

Hemorrhagic shock due to trauma (HS/T) induces an inflammatory response that can contribute to end-organ injury. The pathways involved in the initiation and propagation of HS/T-induced inflammation are incompletely understood. Here, we hypothesized that the DNA sensor TLR9 would have a role in inflammatory signaling after HS/T. Using mice expressing a nonfunctional, mutant form of TLR9, we identified a role of TLR9 in driving the initial cytokine response and liver damage in a model of hemorrhagic shock and bilateral femur fracture. Circulating DNA levels were found to correlate with the degree of tissue damage. Experiments using chimeric mice show that TLR9 on both bone marrow-derived cells and parenchymal cells are important for the TLR9-mediated liver and tissue damage, as well as systemic inflammation after HS/T. These data suggest that release of DNA may be a driver of the inflammatory response to severe injury as well as a marker of the extent of tissue damage. One of the sensors of DNA in the setting of HS/T seems to be TLR9.

The extracellular signal-regulated kinases/mitogen activated protein kinase (ERK/MAPK) and nuclear factor-κB (NF-κB) pathways are critical for cell survival and proliferation. Alpinumisoflavone (AIF), isolated from the African medicinal plant Erythrina lysistemon, is a member of the isoflavone group. In this report, we demonstrated that AIF treatment induces cell death of human lung tumor cells. Incubation of lung tumor cells with AIF increased the sub-G1 population and caspase 3/7 activity, suggesting that the cell death is caused by apoptosis. To identify the signaling pathway involved in the tumor cell death, we examined the modulation of transcriptional activity using various reporter constructs and found that AIF significantly deregulated both the ERK/MAPK and NF-κB pathways. Western blot analysis with antibodies to MAP/ERK kinase (MEK) and ERK showed that AIF dephosphorylates both MEK and ERK. Alpinumisoflavone also repressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells by inhibiting NF-κB-dependent transcription. Therefore, the cell death induced by AIF may be via repressing both the ERK/MAPK and NF-κB pathways.

Classical Hodgkin lymphoma (cHL) is a malignancy of B-cell origin in which the neoplastic cells, known as "Reed-Sternberg" (RS) cells, are characteristically binucleated. Here we describe a family where multiple individuals developing cHL have inherited a reciprocal translocation between chromosomes 2 and 3. The translocation disrupts KLHDC8B, an uncharacterized gene from a region (3p21.31) previously implicated in lymphoma and related malignancies, resulting in its loss of expression. We tested KLHDC8B as a candidate gene for cHL and found that a 5'-UTR polymorphism responsible for decreasing its translational expression is associated with cHL in probands from other families with cHL and segregates with disease in those pedigrees. In one of three informative sporadic cases of cHL, we detected loss of heterozygosity (LOH) for KLHDC8B in RS cells, but not reactive T lymphocytes, purified from a malignant lymph node. KLHDC8B encodes a protein predicted to contain seven kelch repeat domains. KLHDC8B is expressed during mitosis, where it localizes to the midbody structure connecting cells about to separate during cytokinesis, and it is degraded after cell division. Depletion of KLHDC8B through RNA interference leads to an increase in binucleated cells, implicating its reduced expression in the formation of cHL's signature RS cell.

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI.Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation,but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERKactivation and PI3K/Akt/eNOS/NO signaling.

Phospholipase D (PLD) has been implicated in survival and anti-apoptosis, but the molecular mechanism by which it responds to apoptotic stimuli is poorly unknown. Here, we demonstrate that cleavage of PLD isozymes as specific substrates of caspase differentially regulates apoptosis. PLD1 is cleaved at one internal site (DDVD(545)S) and PLD2 is cleaved at two or three sites (PTGD(13)ELD(16)S and DEVD(28)T) in the front of N-terminus. Cleavage of PLD was endogenously detected in post-mortem Alzheimer brain together with activated caspase-3, suggesting the physiological relevance. The cleavage of PLD1 but not PLD2 might act as an inactivating process since PLD1 but not PLD2 activity is significantly decreased during apoptosis, suggesting that differential cleavage of PLD isozymes could affect its enzymatic activity. Moreover, caspase-resistant mutant of PLD1 showed more potent anti-apoptotic capacity than that of wild type PLD1, whereas PLD2 maintained anti-apoptotic potency in spite of its cleavage during apoptosis. Moreover, PLD2 showed more potent anti-apoptotic effect than that of PLD1 in overexpression and knockdown experiments, suggesting that difference in anti-apoptotic potency between PLD1 and PLD2 might be due to its intrinsic protein property. Taken together, our results demonstrate that differential cleavage pattern of PLD isozymes by caspase might affect its enzymatic activity and anti-apoptotic function.

Nitric oxide (NO) plays an important role in maintaining vascular homeostasis. The importance of NO in the vasculature is demonstrated by several experimental conditions, such as vascular endothelial growth factor (VEGF)-induced angiogenesis. Thus, the NO metabolic pathway in endothelial cells could be one of the main contributing factors for angiogenesis. Although several methods have been used for measuring in vitro angiogenesis, a proper technique has not been developed for identifying in vivo NO-dependent angiogenesis. This chapter provides a new intravital microscopic method for detecting and measuring NO-dependent angiogenesis in a mouse model. This technique showed strong abdominal neovascularization in wild-type mice, but not eNOS knockout mice, locally injected with VEGF, as well as stimulation of angiogenesis in NO donor-injected mice. This technique also revealed the inhibitory effect of the NOS inhibitor N(G)-iminoethyl-L-ornithine in VEGF-mediated in vivo angiogenesis. This chapter describes intravital microscopy as a new imaging technique for detecting NO-dependent angiogenesis in an animal model.

Angiogenesis is important for promoting cardiovascular disease, wound healing, and tissue regeneration. We investigated the effects of Korean red ginseng water extract (KRGE) on angiogenesis and its underlying signal mechanism. KRGE increased in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells, as well as stimulated in vivo angiogenesis without increasing VEGF expression. KRGE-induced angiogenesis was accompanied by phosphorylation of ERK1/2, phosphatidylinositol 3-kinase (Akt), and endothelial nitric oxide synthase (eNOS) as well as an increase in NO production. Inhibition of PI3K activity by wortmannin completely inhibited KRGE-induced angiogenesis and phosphorylation of Akt, ERK1/2, and eNOS, indicating that PI3K/Akt activation is an upstream event of the KRGE-mediated angiogenic pathway. The MEK inhibitor PD98059 blocked KRGE-induced ERK1/2 phosphorylation without affecting Akt and eNOS activation. However, the eNOS inhibitor N(G)-monomethyl-L-arginine effectively inhibited tube formation, but partially blocked proliferation and migration as well as ERK phosphorylation, without altering Akt and eNOS activation, revealing that the eNOS/NO pathway is partially involved in ERK1/2 activation. This study demonstrated that KRGE stimulates in vitro and in vivo angiogenesis through the activation of the PI3K/Akt-dependent ERK1/2 and eNOS signal pathways and their cross talk.

Colchicine has been shown to regulate the expression of inflammatory gene, but this compound possesses much weaker anti-inflammatory activity. In this study, we synthesized a new colchicine derivative CT20126 and examined its immunomodulatory property. CT20126 was found to have immunosuppressive effects by inhibiting lymphocyte proliferation without cytotoxicity and effectively inhibit the transcriptional expression of the inflammatory genes, iNOS, TNF-alpha, and IL-1beta, in macrophages stimulated by LPS. This effect was nearly comparable to that of cyclosporine A. This compound also significantly suppressed the production of nitric oxide and Th1-related pro-inflammatory cytokines, IL-1beta, TNF-alpha, and IL-2, with minimal suppression of Th2-related anti-inflammatory cytokines IL-4 and IL-10 in the sponge matrix allograft model. Moreover, administration of CT20126 prolonged the survival of allograft skins from BALB/c mice (H-2(d)) to the dorsum of C57BL/6 (H-2(b)) mice. The in vivo immune suppressive effects of CT20126 were similar to that of cyclosporine A. These results indicate that this compound may have potential therapeutic value for transplantation rejection and other inflammatory diseases.