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KR Jyothi,1 Jagadish Beloor,2 Ara Jo,1 Minh Nam Nguyen,1 Tae Gyu Choi,1 Jin-Hwan Kim,1 Salima Akter,1 Sang-Kyung Lee,2 Chi Hoon Maeng,3 Hyung Hwan Baik,1 Insug Kang,1 Joohun Ha,1 Sung Soo Kim1 1Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul, Republic of Korea; 2Department of Bioengineering and Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul, Republic of Korea; 3Department of Medical Oncology and Hematology, Kyung Hee University Hospital, Seoul, Republic of Korea Abstract: Therapeutic options for hepatitis C virus (HCV) infection have been limited by drug resistance and adverse side effects. Targeting the host factor cyclophilin A (CypA), which is essential for HCV replication, offers a promising strategy for antiviral therapy. However, due to its immunosuppressive activity and severe side effects, clinical application of cyclosporine A (CsA) has been limited as an antiviral agent. To overcome these drawbacks, we have successfully developed a liver-specific, sustained drug delivery system by conjugating the liver-targeting peptide (LTP) to PEGylated CsA-encapsulated poly (lactic-co-glycolic) acid (PLGA) nanoparticles. Furthermore, our delivery system exhibited high specificity to liver, thus contributing to the reduced immunosuppressive effect and toxicity profile of CsA. Finally, targeted nanoparticles were able to effectively inhibit viral replication in vitro and in an HCV mouse model. As a proof of principle, we herein show that our delivery system is able to negate the adverse effects of CsA and produce therapeutic effects in an HCV mouse model.Keywords: HCV, liver-targeting peptide, targeted drug delivery

Diabetes mellitus is a common disease that has affected people since antiquity. The complexity of diabetes mellitus lies in the fact that it requires continuous management in order to prevent serious complications. Various methods for managing diabetes mellitus through monitoring blood glucose levels have been studied and developed, and the most widely used method includes using an invasive blood glucose meter. Since the invasive blood glucose meter poses a major inconvenience for patients, in this study, we sought to develop a non-invasive blood glucose meter. We therefore proposed the development of a non-invasive blood glucose measurement system that is based on near-infrared spectroscopy. The system developed was composed of a light source for emitting different wavelengths, a light detector unit, and a computing system for recording signals. A prepared glucose solution was injected into a quartz cuvette and light at 780-1650 nm was emitted to pass through the cuvette. The degree of reaction was determined by recording the change in wavelength. The wavelength band used for the experiment was 780-1000 nm and the resolution was 20 nm. The glucose concentration was determined to be between 50 to 400 mg/dl compared to the normal range of 80 to 120 mg/dl. By examining which wavelength bands specifically reacted with glucose, we observed that the wavelength bands that decreased or increased in response to the glucose concentration were at 780 nm and 940 nm. For wavelength bands at 1000 nm or above, light was also absorbed by water and therefore it was difficult to distinguish the results. The most reliable wavelength band was at 940 nm with an R ² of 0.9806. In conclusion, the near-infrared light emitting diode based non-invasive glucose detection system performed well and is expected to be a superior method for monitoring blood glucose levels in diabetes mellitus.

Rheumatoid arthritis is a chronic inflammatory disease that affects joints and induces pain and swelling. We evaluated the anti-inflammatory effects of step electrical stimulation (SES) in this study. SES was carried out by increasing the voltage (3 V/s) from 5 V to 100 V for 60 cycles. The viability of mouse embryonic fibroblasts (NIH-3T3) was evaluated after step-electrical stimulation. After the injection of complete Freund's adjuvant (CFA) on the right hind paw of Sprague Dawley (SD) rats (6 weeks old), the degree of swelling was measured using a digital plethysmometer and Vernier caliper. Histological changes in inflamed tissues were observed with hematoxylin and eosin (H&E) staining, while the degree of inflammation was evaluated from the expression level of inflammatory factors such as cyclooxygenase-2 (COX-2), tumor necrosis factor- α (TNF- α ), and interleukin-6 (IL-6). As a result, we found no difference in cell viability after SES treatment between the control and SES-treated groups. On day 21 after CFA injection, the swelling of right hind paws decreased by 1.09 times in SES-treated group as compared with the untreated group. In addition, the levels of COX-2, TNF- α and IL-6 significantly decreased after SES treatment. Thus, SES treatment decreased paw swelling and alleviated inflammation.

Following the publication of the above article and a Corrigendum published in July 2018, the authors have noted an additional error, associated with the presentation of Fig. 1C. Fig 1C showed that β‑N‑methylamino‑L‑alanine induces neuronal apoptotic cell death; however, an error was made in the compilation of this figure and an incorrect band image was selected for α‑actinin, the loading control panel for Fig. 1C. A corrected version of Fig. 1 is shown opposite, incorporating the correct α‑actinin protein bands in Fig. 1C. This change affects neither the interpretation of the data nor conclusions of this work. We regret that this further error went unnoticed at the time, and thank the Editor for allowing us the opportunity to publish this additional Corrigendum. [the original article was published in the Molecular Medicine Reports 14: 4873‑4880, 2016; DOI: 10.3892/mmr.2016.5802].

The present study examined the neuroprotective effects of capsaicin (CAP) and explored their underlying mechanisms in a lipopolysaccharide (LPS)-lesioned inflammatory rat model of Parkinson's dieases (PD). LPS was unilaterally injected into the substantia nigra (SN) in the absence or presence of CAP or capsazepine (CZP, a TRPV1 antagonist). The SN tissues were prepared for immunohistochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, western blot analysis, blood-brain barrier (BBB) permeability evaluation, and reactive oxygen species (ROS) detection. We found that CAP prevented the degeneration of nigral dopamine neurons in a dose-dependent manner and inhibited the expression of proinflammatory mediators in the LPS-lesioned SN. CAP shifted the proinflammatory M1 microglia/macrophage population to an anti-inflammatory M2 state as demonstrated by decreased expression of M1 markers (i.e., inducible nitric oxide synthase; iNOS and interleukin-6) and elevated expression of M2 markers (i.e., arginase 1 and CD206) in the SN. RT-PCR, western blotting, and immunohistochemical analysis demonstrated decreased iNOS expression and increased arginase 1 expression in the CAP-treated LPS-lesioned SN. Peroxynitrate production, reactive oxygen species levels and oxidative damage were reduced in the CAP-treated LPS-lesioned SN. The beneficial effects of CAP were blocked by CZP, indicating TRPV1 involvement. The present data indicate that CAP regulated the M1 and M2 activation states of microglia/macrophage in the LPS-lesioned SN, which resulted in the survival of dopamine neurons. It is therefore likely that TRPV1 activation by CAP has therapeutic potential for treating neurodegenerative diseases, that are associated with neuroinflammation and oxidative stress, such as PD.

PR interval is the period from the onset of P wave to the start of the QRS complex on electrocardiograms. A recent genomewide association study (GWAS) suggested that GAREM1 was linked to the PR interval on electrocardiograms. This study was designed to validate this correlation using additional subjects and examined the function of Garem1 in a mouse model. We analyzed the association of rs17744182, a variant in the GAREM1 locus, with the PR interval in 5646 subjects who were recruited from 2 Korean replication sets, Yangpyeong (n = 2471) and Yonsei (n = 3175), and noted a significant genomewide association by meta-analysis (P = 2.39 × 10 -8 ). To confirm the function of Garem1 in mice, Garem1 siRNA was injected into mouse tail veins to reduce the expression of Garem1. Garem1 transcript levels declined by 53% in the atrium of the heart (P = 0.029), and Garem1-siRNA injected mice experienced a significant decrease in PR interval (43.27 ms vs. 44.89 ms in control, P = 0.007). We analyzed the expression pattern of Garem1 in the heart by immunohistology and observed specific expression of Garem1 in intracardiac ganglia. Garem1 was expressed in most neurons of the ganglion, including cholinergic and adrenergic cells. We have provided evidence that GAREM1 is involved in the PR interval of ECGs. These findings increase our understanding of the regulatory signals of heart rhythm through intracardiac ganglia of the autonomic nervous system and can be used to guide the development of a therapeutic target for heart conditions, such as atrial fibrillation.

Ethnopharmacological Relevance: Cordyceps militaris is an ingredient of traditional Chinese medicine and have been widely used for inflammatory diseases and cancer. Cordycepin is one of the major bioactive components of Cordyceps militaris, and has been known to have anti-inflammatory and anti-oxidant effects., Aim of This Study: In the present study, we examined whether WIB-801C, a standardized and cordycepin-enriched extract of caterpillar fungus (Cordyceps militaris), would attenuate blood-spinal cord barrier (BSCB) disruption by inhibiting matrix metalloprotease (MMP)-9 activity, leading to improvement of functional outcomes after spinal cord injury (SCI)., Materials and Methods: Male Sprague-Dawley rats were subjected to contusive SCI using a New York University (NYU) impactor, and WIB-801C (50mg/kg) was administered at 2h and 8h after injury orally and further treated once a day for indicated time points. BSCB disruption, MMP-9 activity, blood infiltration, inflammation, neuronal apoptosis, axonal loss, demyelination, and neurological deficit were evaluated., Results: We found that WIB-801C significantly attenuated BSCB disruption by inhibiting MMP-9 expression and activation after injury. The infiltration of neutrophils at 1 d and macrophage at 5 d after SCI was also ameliorated by WIB-801C as compared with vehicle control. In addition, the expression of inflammatory cytokines and mediators such as Tnf-α, IL-1β, IL-6, Cox-2, and inos as well as chemokines such as Gro-α and Mip-2α was significantly inhibited by WIB-801C. Furthermore, WIB-801C inhibits p38MAPK activation and proNGF production in microglia after injury. These events eventually led to the inhibition of apoptotic cell death of neurons and oligodendrocytes, improved functional recovery and attenuated demyelination and axon loss after SCI., Conclusion: Our results suggest that WIB-801C can be used as a therapeutic agent after SCI by attenuating BSCB disruption followed inflammation., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

β-N-methylamino-L-alanine (BMAA) is a neurotoxin that is closely associated with the incidence of amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease. In cultured neuronal cells, BMAA notably induces the upregulation of endoplasmic reticulum (ER) chaperons and activates the unfolded protein response (UPR) receptor pathways of protein kinase RNA‑like endoplasmic reticulum kinase, inositol‑requiring kinase 1 and transcription factor 6. The ER stress‑specific protein CCAAT/‑enhancer‑binding protein homologous protein (CHOP) affords pro‑apoptotic responses that cause mitochondrial damage and caspase activation. BMAA also induces the activation of mitogen‑activated protein kinase member c‑JUN N‑terminal kinase, p38 and extracellular signal‑regulated kinase, which have been suggested to be involved in the signaling pathway of UPR‑mediated apoptosis. Inhibition of ER stress using ER stress antagonist, salubrinal, attenuated the expression of CHOP and alleviated neuronal death. Overexpression of heat shock protein 70 suppressed the activation of UPR receptors and UPR‑evoked apoptotic signaling. The present findings demonstrated that ER stress induced by BMAA is the important mediator of neuronal injury and apoptotic death, and suggests development in novel therapeutic strategies for treatment.

The cannabinoid (CB2) receptor type 2 has been proposed to prevent the degeneration of dopamine neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. However, the mechanisms underlying CB2 receptor-mediated neuroprotection in MPTP mice have not been elucidated. The mechanisms underlying CB2 receptor-mediated neuroprotection of dopamine neurons in the substantia nigra (SN) were evaluated in the MPTP mouse model of Parkinson's disease (PD) by immunohistochemical staining (tyrosine hydroxylase, macrophage Ag complex-1, glial fibrillary acidic protein, myeloperoxidase (MPO), and CD3 and CD68), real-time PCR and a fluorescein isothiocyanate-labeled albumin assay. Treatment with the selective CB2 receptor agonist JWH-133 (10 μg kg(-1), intraperitoneal (i.p.)) prevented MPTP-induced degeneration of dopamine neurons in the SN and of their fibers in the striatum. This JWH-133-mediated neuroprotection was associated with the suppression of blood-brain barrier (BBB) damage, astroglial MPO expression, infiltration of peripheral immune cells and production of inducible nitric oxide synthase, proinflammatory cytokines and chemokines by activated microglia. The effects of JWH-133 were mimicked by the non-selective cannabinoid receptor WIN55,212 (10 μg kg(-1), i.p.). The observed neuroprotection and inhibition of glial-mediated neurotoxic events were reversed upon treatment with the selective CB2 receptor antagonist AM630, confirming the involvement of the CB2 receptor. Our results suggest that targeting the cannabinoid system may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with glial activation, BBB disruption and peripheral immune cell infiltration.

The hypothalamic-pituitary-adrenal (HPA) axis has an important role in the pathogenesis of stroke. We investigated whether single nucleotide polymorphisms (SNPs) of melanocortin 2 receptor (MC2R), also known as adrenocorticotropic hormone (ACTH) receptor, were associated with the development of intracerebral hemorrhage (ICH) in Korean population. Two promoter SNPs [rs1893219 (-853A/G) and rs1893220 (-759G/T)] were genotyped in 145 ICH patients and 331 control subjects using direct sequencing. Multiple logistic regression models were used to determine odds ratios, 95% confidence intervals, and p-values. Two SNPs were associated with the development of ICH (rs1893219, p=0.003 in log-additive model, p=0.023 in dominant model, p=0.002 in recessive model; rs1893220, p=0.005 in log-additive model, p=0.021 in dominant model, p=0.003 in recessive model). The frequencies of the G allele of rs1893219 and the T allele of rs1893220 were decreased in ICH group compared to control group (p=0.003 and p=0.004, respectively). The frequencies of the AG and GT haplotypes comprised of rs1893219 and rs1893220 were also significantly different between the ICH and control groups (p=0.0026 and p=0.0034, respectively). These data suggest that the MC2R gene may contribute to the development of ICH., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Alopecia areata (AA) is a common disease, which causes hair loss in humans. AA has a genetically complex inheritance. This study investigated the possible correlations between single nucleotide polymorphisms (SNPs) in the promoter regions of the chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1) and chemokine (C-X-C motif) ligand 2 (CXCL2) genes and the development of AA in the Korean population. Two hundred and thirty-five AA patients and 240 control subjects were recruited. The specific SNPs occurring in the promoter regions of the CXCL1 and CXCL2 genes (rs3117604, -429C/T and rs3806792, -264T/C, respectively) were genotyped. All data obtained was evaluated using the SNPStats, SPSS 18.0, and the Haploview v.4.2 software platforms. The Odd's ratios (OR), 95% confidence intervals (CI), and P values were calculated using multiple logistic regression models. Analyses of the genetic sequences obtained revealed a significant correlation between the two SNPs and the development of AA (rs3117604, P = 0.0009 in co-dominant model 1, P = 0.01 in co-dominant model 2, P = 0.004 in the dominant model, P = 0.005 in the log-additive model, P = 0.012 in allele distribution; rs3806792, P = 0.036 in co-dominant model 2, P = 0.0046 in the log-additive model). The TT and CC haplotypes were also observed to show a significant association with increased risk of AA (TT haplotype, P = 0.0018; CC haplotype, P = 0.0349). Our data suggests that the CXCL1 and CXCL2 genes may be associated with AA susceptibility.

Glycogen synthase kinase 3β (GSK3β) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3β by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3β is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3β directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr(356) residue inhibits GSK3β activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3β on obesity. This result suggests a potential in vivo association between GSK3β and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)(356)-GSK3β was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3β activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3β activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3β., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The mechanisms underlying aggregate formation in age-related neurodegenerative diseases remain not well understood. Here we investigated whether dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) is involved in the formation of regulator of calcineurin 1 (RCAN1) aggregates. We show that RCAN1 self-associates and forms multimers, and that this process is promoted by the Dyrk1A-mediated phosphorylation of RCAN1 at the Thr(192) residue. Transgenic mice that overexpress the Dyrk1A exhibited lower levels of phospho-Thr(192)-RCAN1 in 10-month-old-group compared to littermate controls, when analyzed with soluble hippocampus lysates. These results suggest that the phosphorylation of RCAN1 by Dyrk1A stimulates the formation of insoluble aggregates upon aging., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Stress has a deteriorating effect on hippocampal function. It also contributes to symptom exacerbation in many disease states, including overactive bladder and interstitial cystitis/bladder pain syndrome. We investigated the effects of various types of stresses (restraint, noise, and cold) on short-term memory and apoptosis in relation with corticotropin-releasing factor (CRF) expression., Methods: Rats in the restraint stress group were restrained in a transparent Plexiglas cylinder for 60 minutes twice daily. Rats in the noise stress group were exposed to the 120 dB supersonic machine sound for 60 minutes twice daily. Rats in the cold stress group were placed in a cold chamber at 4℃ for 60 minutes twice daily. Each stress was applied for 10 days. A step-down avoidance test for short-term memory, immunohistochemistry for caspase-3 expression, and western blot analysis for Bax and Bcl-2 expressions were conducted., Results: Latency time was decreased and CRF expression in the hippocampal dentate gyrus and hypothalamic paraventricular nucleus were increased in all of the stress groups. The number of caspase-3-positive cells in the hippocampal dentate gyrus was increased and the expressions of Bax and Bcl2 in the hippocampus were decreased in all of the stress groups., Conclusions: All of the stress groups experienced short-term memory impairment induced by apoptosis in the hippocampus. The present results suggest the possibility that these stresses affecting the impairment of short-term memory may also induce functional lower urinary tract disorders.

Annexin A5 (ANXA5), which is known as a protein with anticoagulative function, may play a role in triglyceride biosynthesis. Triglycerides are involved in lipid and energy metabolism, which are important in the elucidation of obesity. To investigate the association between single-nucleotide polymorphisms (SNPs) of ANXA5 and obesity in a Korean population, 372 participants (213 overweight/obese individuals and 159 control subjects) were enrolled from the Kyung Hee University Medical Center and Keimyung University Dongsan Medical Center. The genotypes of five SNPs (rs12510548, rs4240260, rs3756281, rs13136094 and rs6534313) were evaluated in ANXA5 using the multiple logistic regression analysis with the codominant 1, codominant 2, dominant, recessive and log-additive models. The genotype and allele frequencies of the five investigated SNPs exhibited significant differences between the control and the overweight/obese groups: rs12510548 (P=0.004 in the codominant 2 model, P=0.0019 in the recessive model, P=0.027 in the log-additive model and P=0.026 in allele frequencies); rs4240260 (P=0.002 and Fisher's exact P=0.0006 in the codominant 2 model, P=0.0007 and Fisher's exact P=0.0007 in the recessive model, P=0.020 and Fisher's exact P=0.0019 in the log-additive model and P=0.020 in allele frequencies); rs3756281 (P=0.016 in the codominant 2 model and P=0.0094 in the recessive model); rs13136094 (P=0.0030 and Fisher's exact P=0.0011 in the codominant 2 model, P=0.0012 and Fisher's exact P=0.0013 in the recessive model, P=0.034 and Fisher's exact P=0.0035 in the log-additive model and P=0.024 in allele frequencies); and rs6534313 (P=0.0010 and Fisher's exact P=0.0003 in the codominant 2 model, P=0.0003 and Fisher's exact P=0.0003 in the recessive model, P=0.0075 and Fisher's exact P=0.0010 in the log-additive model and P=0.005 in allele frequencies). Two haplotypes were weakly associated with obesity (GGATG, P=0.037 and CAGCC, P=0.020). Results of the present study suggested that ANXA5 may be associated with the development of obesity in a Korean population.

X-Linked inhibitor of apoptosis (XIAP)-associated factor-1 (XAF1) antagonizes XIAP-mediated caspase inhibition. XAF1 also serves as a tumor-suppressor gene, and loss of XAF1 expression correlates with tumor progression. This study investigated whether XAF1 missense single-nucleotide polymorphisms (SNPs) are associated with the development of papillary thyroid cancer (PTC) and their clinicopathological features in a Korean population. Eighty-nine cases of PTC and 276 controls were enrolled. Two missense SNPs [rs34195599 (Glu85Gly) and rs2271232 (Arg132His)] in XAF1 were genotyped using direct sequencing. The SNPStats, SNPAnalyzer, Helixtree, and Haploview version 4.2 programs were used to evaluate genetic data. Multiple logistic regression models were used to determine odds ratios (ORs), 95% confidence intervals (CIs), and p-values. Missense SNP rs34195599 was weakly-associated with the development of PTC (p=0.046 in genotypic distributions; p=0.048 in allelic distributions). For the clinicopathological features, rs34195599 was strongly related to multifocality [unifocality (A/G, 1.7%) vs. multifocality (A/G, 16.7%), OR=11.44, 95% CI=1.27-103.26, p=0.015 in genotypic distributions] [unifocality (G, 0.8%) vs. multifocality (G, 8.3%), OR=10.64, 95% CI=1.21-93.23, p=0.017 in allelic distributions] and location [one lobe (A/G, 1.6%) vs. both lobes (A/G, 19.2%), OR=15.63, 95% CI=1.62-150.46, p=0.008 in genotypic distributions] [one lobe (G, 0.8%) vs. both lobes (G, 9.6%), OR=13.30, 95% CI=1.51-116.82, p=0.009 in allelic distributions]. Our data suggest that the G allele of rs34195599 of XAF1 may be a risk factor for the clinicopathological features of PTC, especially for multifocality and location (both lobes).

Microcephalin 1 (MCPH1) has a crucial role in the DNA damage response by promoting the expression of checkpoint kinase 1 (CHK1) and breast cancer susceptibility gene 1 (BRCA1). MCPH1 containing BRCT domain has been suggested as a tumor suppressor in breast and ovarian cancers. We analyzed the effect of both protein expression and MCPH1 polymorphisms in breast cancer patients. Low nuclear expression of microcephalin was present in 52.4% of breast cancers and was associated with allele T in rs2912010 (p=0.046). However, cytoplasmic microcephalin expression increased with increasing grade (p=0.010). An association between low nucleus microcephalin expression and allele T was identified in rs2912010 (p=0.046). After data analysis, allele distribution of the MCPH1 polymorphisms was not different between breast cancer patients and healthy controls. But the polymorphism was associated with negative status for ER (rs2912010/C2302T; p=0.032, rs1057090/C2358T; p=0.027, rs2912016/C2494A; p=0.024), and allele T in both rs2912010 and rs1057090 was associated with increasing tumor grade (rs2912010; p=0.040, rs1057090; p=0.043) in breast cancer. We are first to report that association of MCPH1 protein expression and its polymorphisms in breast cancer. The MCPH1 polymorphisms and protein expression were associated with tumorigenesis in breast cancer and may be a useful biomarker for identification of the aggressive types of breast cancer., (Copyright © 2013 Elsevier B.V. All rights reserved.)

DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-β-gal positive until 15 days repair incubation. Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Acupuncture (AP) is currently used worldwide to relieve pain. However, little is known about its mechanisms of action. We found that after spinal cord injury (SCI), AP inhibited the production of superoxide anion (O(2)·), which acted as a modulator for microglial activation, and the analgesic effect of AP was attributed to its anti-microglial activating action. Direct injection of a ROS scavenger inhibited SCI-induced NP. After contusion injury which induces the below-level neuropathic pain (NP), Shuigou and Yanglingquan acupoints were applied. AP relieved mechanical allodynia and thermal hyperalgesia, while vehicle and simulated AP did not. AP also decreased the proportion of activated microglia, and inhibited both p38MAPK and ERK activation in microglia at the L4-5. Also, the level of prostaglandin E(2) (PGE2), which is produced via ERK signaling and mediates the below-level pain through PGE2 receptor, was reduced by AP. Injection of p38MAPK or ERK inhibitors attenuated NP and decreased PGE2 production. Furthermore, ROS produced after injury-induced p38MAPK and ERK activation in microglia, and mediated mechanical allodynia and thermal hyperalgesia, which were inhibited by AP or a ROS scavenger. AP also inhibited the expression of inflammatory mediators. Therefore, our results suggest that the analgesic effect of AP may be partly mediated by inhibiting ROS-induced microglial activation and inflammatory responses after SCI and provide the possibility that AP can be used effectively as a non-pharmacological intervention for SCI-induced chronic NP in patients., (Copyright © 2012. Published by Elsevier Inc.)

Aims: The present study examined whether Aβ(1-42) can induce endogenous expression of interleukin-13 (IL-13) or (IL-4) within activated microglia in the rat hippocampus in vivo. We further investigated whether these cytokines mediate ROS/RNS generation through activation of NADPH oxidase and/or inducible nitric oxide synthase (iNOS), and thus contribute to the degeneration of hippocampal neurons in vivo., Results: Here, we show that IL-13 and IL-4, endogenously expressed in Aβ(1-42)-activated microglia in hippocampus in vivo, contribute to degeneration of hippocampal neurons in vivo. Neutralization of IL-13 and IL-4 protected hippocampal neurons in vivo against neurotoxicity by inhibiting activation of microglial NADPH oxidase and iNOS, resulting in attenuation of ROS generation and oxidative damage of protein, lipid and DNA., Innovation: To our knowledge, this is the first study to demonstrate the possible involvement of endogenously expressed IL-13 and/or IL-4 in activated microglia after Aβ(1-42) injection in the degeneration of hippocampal neurons in vivo. The current findings suggest that the deleterious effects of microglia-derived endogenous IL-13 and/or IL-4 are involved in oxidative stress-mediated neurodegenerative diseases, such as AD., Conclusion: We carefully hypothesize that IL-13 and IL-4, well-known as anti-inflammatory cytokines might serve as neurotoxic mediators by enhancing microglia-derived oxidative stress in Aβ(1-42)-treated hippocampus in vivo.

We investigated the effects of synthetic cannabinoids, WIN55,212-2 and HU210, on LPS-injected rat substantia nigra in vivo. Intranigral injection of LPS resulted in a significant loss of nigral dopaminergic (DA) neurons, as determined by Nissl staining and TH immunohistochemistry. LPS-induced neurotoxicity was accompanied by microglial activation, as demonstrated by OX-42 immunohistochemistry. In parallel, Western blot analysis, ELISA assay and hydroethidine histochemistry revealed activation of NADPH oxidase, as demonstrated by increased translocation of the cytosolic proteins p47(phox) and p67(phox), generation of reactive oxygen species (ROS) and increased level of proinflammatory cytokines (TNF-α and IL-1β), where degeneration of nigral DA neurons was evident. Interestingly, WIN55,212-2 and HU210 increased the survival of nigral DA neurons at 7days post-LPS treatment. Consistent with these results, cannabinoids inhibited activation of NADPH oxidase, ROS production and production of proinflammatory cytokines in the rat SN. The present data suggest that cannabinoids may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglial activation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

To investigate whether single nucleotide polymorphisms (SNPs) of eicosanoid biosynthesis genes are associated with intracerebral hemorrhage (ICH) and ischemic stroke (IS), seven SNPs in the coding or promoter regions were selected: ALOX12 (rs434473, Asn322Ser), ALOX5 (rs2228064, Thr90Thr), ALOX5AP (rs17222919, -1316T/G), PTGES (rs7872802, -404A/G), PTGIS (rs5628, Leu256Leu), PTGS1 (rs3842788, Gln41Gln) and PTGS2 (rs5275, 3'UTR). A total of 398 control subjects and 196 stroke patients (79 ICH and 117 IS) were genotyped by direct sequencing. The rs17222919 SNP was associated with ICH in codominant 1 (P=0.008), dominant (P=0.003) and log-additive (P=0.004) models. Allele frequencies of rs17222919 were different between ICH and controls (P=0.007). However, the seven tested SNPs were not associated with clinical phenotypes (NIHSS, MBI and CRPS) in ICH and IS. These results suggest that the promoter SNP rs17222919 of ALOX5AP may be associated with the development of ICH in Korean population., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Transforming growth factor, beta receptor II (TGFBR2) is mainly expressed by neurons in the central nervous system, and reduced neuronal TGFBR2 signaling results in accelerated age-dependent neurodegeneration. To investigate whether TGFBR2 polymorphisms are associated with ischemic stroke (IS) and intracerebral hemorrhage (ICH), two single nucleotide polymorphisms (SNPs) of TGFBR2 gene (rs764522, -1444C/G; rs2228048, Asn389Asn) were selected and genotyped by direct sequencing in 247 stroke patients (120 IS and 127 ICH) and 655 control subjects (260 for IS and 395 for ICH). SNPStats, SNPAnalyzer, Helixtree, and Haploview version 4.2 were used to analyze genetic data. Multiple logistic regression models (codominant, dominant, recessive, and log-additive) were performed to evaluate odds ratios (ORs), 95% confidence intervals (CIs), and p values. The synonymous SNP rs2228048 was significantly associated with ICH (p = 0.032 in codominant 2 model, p = 0.024 in dominant model, p = 0.020 in recessive model, and p = 0.005 in log-additive model) and Fisher's exact test (p = 0.009). Allele frequencies of rs2228048 were different between ICH and controls (p = 0.006). In Bonferroni correction, these correlations were also significant. These results suggest that the synonymous SNP rs2228048 of TGFBR2 gene may be associated with development of ICH in Korean population.

Lipopolysaccharide (LPS)-induced microglial activation causes degeneration of nigral dopaminergic (DA) neurons. Here, we examined whether fluoxetine prevents LPS-induced degeneration of DA in the rat substantia nigra (SN) in vivo. Seven days after LPS injection into the SN, immunostaining for tyrosine hydroxylase (TH) revealed a significant loss of nigral DA neurons. Parallel activation of microglia (visualized by OX-42 and ED1 immunohistochemistry), production of reactive oxygen species (ROS) (assessed by hydroethidine histochemistry), and degeneration of nigral DA neurons were also observed in the SN. Western blot analyses and double-label immunohistochemistry showed an increase in the expression of inducible nitric oxide synthase (iNOS) within activated microglia. LPS also induced translocation of p67(phox), the cytosolic component of NADPH oxidase, to the membrane of SN microglia, indicating activation of NADPH oxidase. The LPS-induced loss of nigral DA neurons was partially inhibited by fluoxetine, and the observed neuroprotective effects were associated with fluoxetine-mediated suppression of microglial NADPH oxidase activation and iNOS upregulation, and decreased ROS generation and oxidative stress. These results suggest that fluoxetine and analogs thereof may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglia-derived oxidative damage., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Gangliosides, sialic acid-containing glycosphingolipids exist in mammalian cell membranes particularly neuronal membranes. The trisialoganglioside (GT1b) is one of the major brain gangliosides and acts as an endogenous regulator in the brain. We previously showed GT1b induces mesencephalic dopaminergic (DA) neuronal death, both in vivo and in vitro. We further investigate the underlying mechanisms of GT1b neurotoxicity., Results: Consistent with earlier findings, GT1b attenuated the DA neuron number and dopamine uptake level in mesencephalic cultures. Morphological evidence revealed GT1b-induced chromatin condensation and nuclear fragmentation as well as an increased number of TUNEL-positive cells, compared to control cultures. Interestingly, while GT1b enhanced caspase-3 activity, DEVD, a caspase-3 inhibitor, failed to rescue DA neuronal death. Immunoblot analysis revealed that GT1b inactivates Akt through dephosphorylation at both Ser473 and Thr308, subsequent dephosphorylation of GSK-3beta, a substrate of Akt, and hyperphosphorylation of tau, downstream of GSK-3beta. Moreover, a GSK-3beta specific inhibitor, L803-mt, attenuated tau phosphorylation and rescued DA neurons from cell death in mesencephalic cultures., Conclusion: Our data provide novel evidence that a Akt/GSK-3beta/tau-dependent, but not caspase-3 signaling pathway plays a pivotal role in GT1b-mediated neurotoxic actions on mesencephalic DA neurons.

We have shown that prothrombin kringle-2 (pKr-2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr-2-induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr-2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr-2. In parallel, pKr-2-activated microglia were detected in the SN with OX-42 and OX-6 immunohistochemistry. Reverse transcription PCR and double-label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and several proinflammatory cytokines. The pKr-2-induced loss of SN DA neurons was partially inhibited by the NOS inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride, and the COX-2 inhibitor DuP-697. Extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were activated in the SN as early as 1 hr after pKr-2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX-2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of mesencephalic neurons and microglia, but not microglia-free neuron-enriched mesencephalic cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr-2, are implicated in the DA neuronal cell death in the SN., ((c) 2009 Wiley-Liss, Inc.)

Although 5-fluorouracil (5-FU) is a widely used chemotherapeutic agent in the treatment of gastric cancer, the underlying mechanism for 5-FU resistant phenotype, has yet to be elucidated. We hypothesized that the sensitivity of gastric cancer to 5-FU treatment might be related to the rate of glucose transport (GLUT), and investigated the expressions of GLUT1, 2, 3, and 4 in two different gastric cancer cells (SNU-216, moderately differentiated gastric adenocarcinoma; and SNU-668, signet ring cell gastric carcinoma). Immunohistochemistry of GLUT1 and GLUT4 and immunoblot analysis of glycogen synthase kinase 3 were also performed. Hexokinase activity was measured. We found that 5-FU suppressed glucose uptake in SNU-216, while it stimulated GLUT in SNU-668. Further analysis revealed that 5-FU decreased the expression levels of GLUT1, 2, and 4 in SNU-216 cells and increased the expression levels of GLUT1, 2, and 4 in SNU-668 cells. Consistent with GLUT expression levels, immunohistochemistry analysis showed that 5-FU increased GLUT1 and GLUT4 levels in SNU-216 and decreased GLUT1 and GLUT4 levels in SNU-668. We also observed that glycogen synthase kinase 3 activity was decreased in SNU-216 and increased in SNU-668 with 5-FU treatment. No significant difference in hexokinase activities was observed with 5-FU treatment. Taken together, these results suggest that 5-FU exerts differential effects on GLUT depending on gastric cancer cell types, which may indicate a possible explanation, at least in part, for the differing responses to 5-FU chemotherapy in gastric cancer.

Stressful experiences, such as an unsatisfactory mother-infant relationship after delivery, can induce depressive disorders, and it is well-known that stressors impair memory function. The hippocampus plays a crucial role in memory processes. In the present study, we determined whether a depressed-like state induced by repeated separation of pups affects the memory capability of the maternal rats. We also determined the effects of repeated separation from pups on cell proliferation, apoptosis, and serotonin expression in the brains of maternal rats. In the present results, the immobility time in the forced swim test was increased and the climbing time was decreased in the mothers separated from their pups. The latency in the step-down avoidance task was increased in the mothers separated from their pups. Also, the expressions of serotonin (5-hydroxytryptamine) and tryptophan hydroxylase in the dorsal raphe were decreased in the mothers separated from their pups. The number of Ki-67-positive cells was decreased, while the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells in the hippocampal dentate gyrus was increased in the mothers separated from their pups. Based on the present results, it is suggested that separation of pups might induce a depressed-like state in the maternal rats with reduced cell proliferation and increased apoptosis in the hippocampus, resulting in memory impairment of maternal rats., ((c) 2009 Elsevier Ireland Ltd. All rights reserved.)

High concentration of epidermal growth factor (EGF) is found in the synovial fluid of rheumatoid arthritis (RA) that might imply the involvement of EGF in the pathogenesis of arthritic diseases. In order to investigate if EGF is involved in the regulation of cyclooxygenase-2 (COX-2) and the prostaglandin E(2) (PGE(2)) production in fibroblast like synoviocytes (FLS) from patients with RA. The levels of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) were evaluated using RT-PCR and Western blot analysis. Electrophoretic mobility shift assay (EMSA) was performed to investigate EGF mediated DNA binding activity of nuclear factor-kappaB (NF-kappaB). PGE(2) levels were analyzed by ELISA. EGF enhanced both COX-2 protein and mRNA expressions. mPGES-1 mRNA level was also increased by EGF treatment. EGF also stimulated ERK1/2 MAPK activity and the inhibition of ERK1/2 by PD098059 (ERK1/2 specific inhibitor) resulted in the suppression of EGF-induced COX-2 expression. The DNA binding activity of NF-kappaB was remarkably increased by EGF treatment and the pretreatment of PD098059 abolished EGF-stimulated NF-kappaB activity. We also observed that the level of PGE(2) was significantly elevated with the treatment of EGF in FLS, and the pretreatment of PD098059 abolished this stimulating effect. These results suggest that EGF is involved in the inflammatory process of RA by stimulating COX-2 expression and PGE(2) production. And EGF enhanced PGE(2) production appears to be mediated via ERK1/2 MAPK and NF-kappaB pathway in FLS.

In the present study, we investigated the effects of IL-13, a well-known anti-inflammatory cytokine, on the thrombin-treated hippocampus in vivo. NeuN immunohistochemistry and Nissl staining revealed significant loss of hippocampal CA1 neurons upon intrahippocampal injection of thrombin. This neurotoxicity was accompanied by substantial microglial activation, as evident from OX-42 immunohistochemistry results. In parallel, Western blot analysis and hydroethidine histochemistry disclosed activation of NADPH oxidase, generation of reactive oxygen species, and oxidative damage in the hippocampal CA1 area showing hippocampal neuron degeneration. Interestingly, immunohistochemical and biochemical experiments showed that intrahippocampal injection of thrombin increased IL-13 immunoreactivity and IL-13 levels as early as 8 h after thrombin, reaching a peak at 7 days, which was maintained up to 14 days. Moreover, double-label immunohistochemistry revealed IL-13 immunoreactivity exclusively in activated microglia. IL-13-neutralizing Abs significantly rescued CA1 hippocampal neurons from thrombin neurotoxicity. In parallel, neutralization of IL-13 inhibited activation of NADPH oxidase, reactive oxygen species production, and oxidative damage. Additionally, IL-13 neutralization suppressed the expression of inducible NO synthase and several proinflammatory cytokines. To our knowledge, the present study is the first to show that IL-13 triggers microglial NADPH oxidase-derived oxidative stress, leading to the degeneration of hippocampal neurons in vivo, as occurs in cases of Alzheimer's disease.

Aromatase encoding by the CYP19 gene catalyzes the conversion of androgens to estrogens. In order to determine if polymorphisms of the CYP19 gene are associated with breast cancer risk, we analyzed the frequency of tetranucleotide (TTTA) tandem repeats and a 3-bp insertion (I)/deletion (D) polymorphism in intron 4 of the CYP19 gene in genomic DNA from 70 Korean breast cancer patients and 102 age-matched, healthy women. The 3-bp deletion allele was found more frequently in the breast cancer group than in the control group (p=0.001). Logistic regression analysis of the CYP 19 insertion/deletion (I/D) genotype showed a strong association between ID polymorphisms and breast cancer. The frequency of DD and ID alleles was significantly increased in the breast cancer group (DD genotype p=0.004, OR=12.81; and ID genotype p=0.005, OR=2.62). However, there were no differences in the genotype distributions of the (TTTA)n polymorphism of CYP19 between breast cancer patients and healthy controls. A positive association was noted between TTTA polymorphisms with 10 or more repeats and ER-negative tumors, as well as between lower repeat polymorphisms and ER-positive tumors (p=0.019). With respect to TTTA polymorphisms, we confirmed that the expression of aromatase in ER-positive MCF7 cells with 7-3 and 11 allele heterozygosity was significantly higher than in ER-negative MDA-MB231 cells with 11 allele homozygosity. These results suggest that 3-bp I/D polymorphisms of the CYP19 gene may be associated with breast cancer and that the (TTTA)n repeat genotype would be useful in selecting candidates for tamoxifen therapy, as well as predicting breast cancer risk in Korean women.

The excessive proliferation and migration of synoviocytes are well-characterized phenomena that play key roles in the pathophysiology of rheumatoid arthritis (RA). Melatonin has been shown to have potent anti-proliferative effect in various cancer cells such as breast and prostate cancer cells. In this study, we examined the role of melatonin on synoviocyte proliferation in primary cultured human fibroblast-like synoviocytes (FLSs) by analyzing protein expression of P21(CIP1) (P21) and P27(KIP1) (P27), the cyclin-dependent kinase inhibitors that are important in cell cycle control, and the phosphorylation of mitogen-activated protein kinases (MAPKs). RA-FLS proliferation was determined by a [(3)H]-thymidine incorporation assay. Western blot analysis was applied to examine the underlying mechanisms of melatonin's effect. Melatonin inhibited RA-FLS proliferation in a dose-dependent manner. It reduced proliferation of passage 2 FLSs by 25% at 10 microm and by nearly 40% at 100 microm concentrations. The inhibitory effect of melatonin on RA-FLS proliferation was also observed in passages 4 and 6. Melatonin upregulated the expression levels of P21 and P27 dose-dependently (24 hr), induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) time-dependently (10 microm), but did not affect phosphorylation of P38 in RA-FLSs. In addition, the expression of P21 and P27 triggered by melatonin was inhibited by the pretreatment of the ERK inhibitor, PD98059 (10 microm). The anti-proliferative action of melatonin in RA-FLSs was also blocked by PD98059. Taken together, these results suggest that melatonin exerts the inhibitory effect of the proliferation of RA-FLSs through the activation of P21 and P27 mediated by ERK. Hence we suggest that melatonin could be used as a therapeutic agent for the treatment of RA.

We investigated the effects of interleukin-4 (IL-4), a well-known anti-inflammatory cytokine, on thrombin-treated rat hippocampi in vivo. Intrahippocampal injection of thrombin resulted in a significant loss of hippocampal CA1 neurons, as determined by Nissl staining and NeuN immunohistochemistry. Thrombin-induced neurotoxicity was accompanied by substantial microglial activation, as demonstrated by OX-42 immunohistochemistry. In parallel, Western blot analysis and hydroethidine histochemistry revealed activation of NADPH oxidase (as demonstrated by increased translocation of the cytosolic proteins p67(phox) and p47(phox)), generation of reactive oxygen species (ROS), and oxidative damage in the hippocampal CA1 area, where degeneration of hippocampal neurons was evident. Interestingly, immunohistochemical and biochemical analysis demonstrated that intrahippocampal injection of thrombin increased immunoreactivity and levels of IL-4 as early as 8 h post-treatment, reaching a peak at 7 days that was maintained for up to 14 days. Moreover, double-label immunohistochemistry detected IL-4 immunoreactivity solely in activated microglia. In experiments to explore the involvement of IL-4 in neurotoxicity, IL-4-neutralizing antibodies significantly increased the survival of CA1 hippocampal neurons at 7 days post-thrombin treatment. Consistent with these results, IL-4 neutralization inhibited activation of NADPH oxidase, ROS production and oxidative damage. Thus, the present study is the first to demonstrate that IL-4 generates microglial NADPH oxidase-derived oxidative stress and leads to the degeneration of hippocampal neurons in vivo, as occurs in Alzheimer's disease.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) beta. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPKalpha1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-kappaB) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-kappaB activation.

Hypoxia-inducible factor-1 (HIF-1), the key transcription factor of hypoxia-inducible genes, is known to be involved in inflammation and immune response, but little is known about the regulation of HIF-1 during microglial activation. Thus, we examined effect of lipopolysaccharide (LPS) on HIF-1 activation and its signaling mechanism in BV2 microglial cells. LPS induced HIF-1alpha mRNA and protein expression as well as HIF-1 transcriptional activation. Moreover, HIF-1alpha knockdown by small interfering RNA (siRNA) decreased LPS-induced expression of hypoxia responsive genes, VEGF, iNOS, and COX-2. We then showed that LPS-induced HIF-1alpha mRNA expression was blocked by an antioxidant, NADPH oxidase inhibitors, and siRNA of gp91phox, a subunit of NADPH oxidase. In addition, we showed that specific pharmacological inhibitors of PI 3-kinase and protein kinase C decreased LPS-induced HIF-1alpha mRNA expression. Finally, we showed that inhibition of transcription factor Sp1 by mithramycin A or Sp1 siRNA decreased LPS-induced HIF-1alpha mRNA and protein expression. Consistently, LPS increased Sp1 DNA binding and its transcriptional activity. Taken together, these results suggest that LPS induces HIF-1alpha mRNA expression and activation via NADPH oxidase and Sp1 in BV2 microglia.

Melatonin possesses a number of important biologic activities including oncostatic, anti-oxidant, and immunostimulatory actions. This study was designed to assess the effects of melatonin on inflammation-related gene expression in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs), using CombiMatrix 2K Human Inflammation chip. After pretreatment with melatonin (100 microm) for 4 hr, cells were incubated with LPS (1 microg/mL) for 24 hr. We compared gene expression profiles between LPS-treated, melatonin-treated, LSP/melatonin-treated, and control groups. LPS induced the upregulation of 95 genes, compared with controls. Melatonin pretreatment in LPS-stimulated PBMCs suppressed the expression of 23 genes more than twofold. Interestingly, melatonin showed a suppressive effect on the expression of CC chemokine subfamily genes, including CCL2/MCP1, CCL3/MIP1 alpha, CCL4/MIP1 beta, CCL5/RANTES, CCL8/MCP2, CCL20/MDC, and CCL22/MIP3 alpha, in LPS-stimulated PBMCs. This result was confirmed by reverse transcriptase polymerase chain reaction. Among the CC chemokine subfamily genes, particularly, the expression of CCL2 and CCL5 was markedly downregulated by melatonin in LPS-stimulated PBMCs. The secretion levels of CCL2 and CCL5 were measured using enzyme-linked immunosorbent assay. Stimulation of PBMCs by LPS induced the secretion of CCL2 (2334.3 +/- 161.4 pg/mL, mean +/- S.E.M.), whereas melatonin pretreatment (153.0 +/- 3.8 pg/mL) inhibited the LPS-induced secretion of CCL2. Melatonin pretreatment (2696.2 +/- 385.3 pg/mL) also inhibited the LPS-induced secretion of CCL5 (4679.6 +/- 107.5 pg/mL). Taken together, these results suggest that melatonin may have a suppressive effect on LPS-induced expression of CC chemokine genes, especially CCL2 and CCL5, which may explain its beneficial effects in the treatment of various inflammatory conditions.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis and its activation during T cell receptor stimulation has recently been reported. In this study, we examined the role of AMPK in interleukin (IL)-2 production in T cells. Inhibition of AMPK by compound C, a specific inhibitor of AMPK or small interfering RNA of AMPKalpha1 suppressed IL-2 production in Jurkat T cells and peripheral blood lymphocytes stimulated with PMA plus ionomycin (PMA/Io) or with monoclonal anti-CD3 plus anti-CD28. We then showed that AMPK inhibition reduced PMA/Io-induced IL-2 mRNA expression and IL-2 promoter activation. Moreover, inhibition of AMPK suppressed transcriptional activation of NF-AT and AP-1, but not NF-kappaB, in PMA/Io-activated Jurkat cells. Finally, we found that compound C inhibited PMA/Io-induced phosphorylation of p38, JNK, and GSK-3beta but not of ERK. These results suggest that AMPK mediates IL-2 production by regulating NF-AT and AP-1activation during T cell stimulation.

Context: Very recently the unforeseen role of syndecan 3 (SDC3), a family of membrane-bound heparin sulfate proteoglycans, in the regulation of energy balance has been discovered in the Sdc3 null female mice., Objective: The objective of the study was to test the hypothesis that single nucleotide polymorphisms (SNPs) in SDC3 are associated with obesity in the Korean population., Design/setting/subjects: We conducted a population-based cohort study consisting of 229 control and 245 study subjects and a second independent study consisting of 192 control and 115 study subjects., Main Outcome Measurement: Body mass index (BMI) was measured., Results: First, Sdc3 mRNA expression in the brain of ob/ob mice was profoundly increased, compared with control mice. Next, all three nonsynonymous SNPs [T271I (rs2282440, C>T), D245N (rs4949184, C>T), and V150I (rs2491132, C>T)] in the SDC3 gene in control female subjects (BMI < 23, n = 229) and obese female subjects (BMI > 30, n = 245) were genotyped. We demonstrated the presence of clear ethnic differences in three nonsynonymous SDC3 SNPs among African-Americans, Chinese, Europeans, and Koreans. Of three SNPs in SDC3, rs4949184 was not associated with obesity and the other two SNPs (rs2282440 and rs2491132) were strongly associated with obesity (P < 0.0001), and the results were confirmed in the second independent study group. Haplotype analysis also revealed strong association with obesity (chi2 = 76.92, P < 0.000001)., Conclusions: There are ethnic differences in the SDC3 polymorphisms, and the polymorphisms are strongly associated with obesity.

The use of topiramate (TPM) in the treatment of binge-eating disorder, bulimia nervosa, and antipsychotic-induced weight gain has recently increased, however, the exact molecular basis for its effects on body weight reduction and improved glucose homeostasis, is yet to be elucidated. Here we investigated the effect and signaling pathway of TPM on glucose uptake in L6 rat skeletal muscle cells, which account for >70% of glucose disposal in the body. Intriguingly, we found that TPM (10 microM) stimulated the rate of glucose uptake up to twofold increase. And TPM-stimulated glucose transport was inhibited with the overexpression of dominant-negative form of AMP-activated protein kinase (AMPK), an important mediator in glucose transport, implicating that AMPK-mediated pathway is involved. The TPM-stimulated glucose transport was blocked by SB203580, a specific inhibitor of AMPK downstream mediator, p38 mitogen-activated protein kinase (MAPK) protein. LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, which is another crucial mediator in independent glucose transport pathway, did not inhibit TPM-stimulated glucose transport. We also found that TPM increased the phosphorylation level of AMPK and p38 MAPK, whereas no effect on the activity of PI 3-kinase of TPM, when assessed by PI 3-kinase assay, was observed. These results together suggest that TPM stimulates glucose transport, not via PI 3-kinase mediated, but via AMPK-mediated pathway in skeletal muscle cells, thereby contributing to the body weight regulation and glucose homeostasis.

The prevalence of diabetes has exponentially increased in recent decades due to environmental factors such as nocturnal lifestyle and aging, both of which influence the amount of melatonin produced in the pineal gland. The present study investigated the effect of melatonin on signaling pathways of glucose transport in C2C12 mouse skeletal muscle cells. Intriguingly, treatment of C2C12 cells with melatonin (1 nm) stimulated glucose uptake twofold increase. Melatonin-stimulated glucose transport was inhibited with co-treatment with the melatonin receptor antagonist luzindole. Furthermore, treatment of stably over-expressed melatonin receptor type 2B containing C2C12 myotubes with melatonin amplified glucose transport c. 13-fold. Melatonin also increased the phosphorylation level of insulin receptor substrate-1 (IRS-1) and the activity of phosphoinositide 3-kinase (PI-3-kinase). However, 3',5'-cyclic adenosine monophosphate-activated protein kinase (AMPK), another important glucose transport stimulatory mediator via an insulin-independent pathway, was not influenced by melatonin treatment. Activity of p38 mitogen-activated protein kinase (MAPK), a downstream mediator of AMPK, was also not changed by melatonin. In addition, melatonin increased the expression level of forkhead box A2, which was recently discovered to regulate fatty acid oxidation and to be inhibited by insulin. In summary, melatonin stimulates glucose transport to skeletal muscle cells via IRS-1/PI-3-kinase pathway, which implies, at the molecular level, its role in glucose homeostasis and possibly in diabetes. Additionally, exposure to light at night and aging, both of which lower endogenous melatonin levels may contribute to the incidence and/or development of diabetes.

It is well documented that manganese neurotoxicity induces clinical symptoms similar to those of idiopathic Parkinson's disease. Although microglial cytotoxic mediator-induced neurotoxicity is suggested, the mechanism by which manganese up-regulates cytotoxic mediator, such as nitric oxide (NO), remains poorly understood. Therefore, in this study, we investigated the mechanism of manganese on induction of iNOS in microglial cells. iNOS promoter/luciferase assay revealed that manganese (500 (M) regulated the iNOS expression at the transcriptional level. Immunoblot analysis also revealed that phosphorylation levels of ERK, JNK MAPKs and Akt (PKB, PI 3-kinase downstream effector), were increased. Both protein and mRNA levels of iNOS expression were abrogated by specific inhibitors, SP600125 (JNK inhibitor, 20 microM), PD98059 (ERKs inhibitor, 50 microM), or LY294002 (PI 3-kinase inhibitor, 20 microM), but not by SB203580 (20 microM), a p38 specific inhibitor. These data lead to the conclusion that manganese regulates the iNOS expression at the transcriptional level in BV2 microglial cells and the increased iNOS protein expression is mediated via both JNK-ERK MAPK and PI3K/Akt signaling pathways, but not via p38 MAPK pathway. Increased iNOS protein level was also found in RAW264.7 murine macrophage cells.

The existence of specific melatonin-binding sites in lymphoid cells led to the discovery of signal transduction pathway for melatonin in human lymphocytes and immunomodulatory role of melatonin in immune cells. In recent years, transcriptional regulation of melatonin on various transcription factors has been demonstrated. Therefore, this study was designed to assess by cDNA microarray analysis the regulatory effects of melatonin on transcription factors in human peripheral blood mononuclear cells (PBMCs). Forty-six genes were upregulated and 23 were downregulated more than twofold in melatonin-treated PBMCs. Of the more than twofold upregulated transcription factor genes, homeo box A4 (HOXA4), forkhead box O1A (FOXO1A), transcription elongation factor B (SIII), polypeptide 3 (TCEB3), and peroxisome proliferative activated receptor delta (PPARD) were identified. Of the more than twofold downregulated genes, PHD finger protein 15 (PHF15) and zinc finger protein 33a (ZNF33A) were identified. In summary, identification of these genes by cDNA microarray analysis in response to melatonin administration may provide a foundation for further studies on the function of melatonin in human PBMCs.

A body of recent evidence implicates that antidepressants affect the inflammatory response and immune system. The present study is focused on the effects of the most widely used antidepressant agent, fluoxetine on the production of nitric oxide (NO) in BV2 microglial cells. In this study, we observed interesting result that NO production was increased by fluoxetine. The mRNA level of nitric oxide synthase (iNos, Nos2) by RT-PCR was also stimulated by fluoxetine. We next conducted electophoretic mobility shift assay (EMSA) to determine the DNA binding activity of nuclear factor kappa B (Nfkappab), an important upstream modulator for Nos2 expression, to find that fluoxetine increased DNA binding activity of Nfkappab. By Western blot analysis, phosphorylation levels of p38 mitogen-activated protein kinase (p38 Mapk, Mapk14) and extracellular signal-related kinase (Erk)1/2 Mapk, upstream signaling mediators of Nfkappab were found to be increased by fluoxetine. In addition, the mRNA expressions of other proinflammatory cytokines, interleukin 6 (Il6) and tumor necrosis factor alpha (Tnfalpha) were examined. The expressions of both Il6 and Tnfalpha by fluoxetine treatment were similar to those of Nos2 and Nfkappab. Taken together, our results show that fluoxetine stimulates NO production via Nfkappab-mediated pathway in BV2 cells.

Adenosine is an endogenous nucleoside that regulates many processes, including inflammatory responses, through activation of its receptors. Adenosine receptors have been reported to be expressed in microglia, which are major immune cells of brain, yet little is known about the role of adenosine receptors in microglial cytokine production. Thus, we investigated the effect of adenosine and adenosine A3 receptor ligands on LPS-induced tumor necrosis factor (TNF-alpha) production and its molecular mechanism in mouse BV2 microglial cells. Adenosine and Cl-IB-MECA, a specific adenosine A3 receptor agonist, suppressed LPS-induced TNF-alpha protein and mRNA levels. Moreover, MRS1523, a selective A3 receptor antagonist, blocked suppressive effects of both adenosine and Cl-IB-MECA on TNF-alpha. We further examined the effect of adenosine on signaling molecules, such as PI 3-kinase, Akt, p38, ERK1/2, and NF-kappaB, which are involved in the regulation of inflammatory responses. Adenosine inhibited LPS-induced phosphatidylinositol (PI) 3-kinase activation and Akt phosphorylation, whereas it had no effect on the phosphorylation of p38 and ERK1/2. We also found that adenosine as well as Cl-IB-MECA inhibited LPS-induced NF-kappaB DNA binding and luciferase reporter activity. Taken together, these results suggest that adenosine A3 receptor activation suppresses TNF-alpha production by inhibiting PI 3-kinase/Akt and NF-kappaB activation in LPS-treated BV2 microglial cells.

The interleukin 4 receptor (IL4R) polymorphism Q576R (rs 180275) has been well known to be associated with atopy and other inflammatory diseases. A single nucleotide polymorphism (SNP) A > G transition potentiates the binding specificity of the adjacent tyrosine residue. In this study we investigated the possible relationship between sudden deafness (SD) and IL4R polymorphism Q576R in 97 Korean SD patients and 613 controls using pyrosequencing method. The odds ratio (OR) for SD associated with the G vs. A allele was 2.58 [P < 0.0001, 95% confidence interval (CI) = 1.84-3.60]. We then sub-grouped SD into Tinnitus positive (+) and Tinnitus negative (-). G allele in Tinnitus (+) is significantly associated with the development of Tinnitus (+) [X(2) = 32.02, P < 0.0001, OR (95% CI) = 2.74 (1.91-3.93)] but not with Tinnitus (-). Taken together these results suggest that G allele could be a risk factor for SD.

Melatonin is reported to be an anti-inflammatory agent. No genetic study concerning the association between melatonin and inflammatory disease has yet been reported. Here we performed a polymorphism study on the melatonin receptor type 1B (MTNR1B) in Korean rheumatoid arthritis (RA) patients and controls. The polymorphism of MTNR1B located in 3'-untranslated region (rs 1562444) was selected for its higher rate of heterozygosity among other single nucleotide polymorphisms (SNPs) in both MTNR1A and MTNR1B genes and investigated in RA patients (n = 173) and healthy controls (n = 195) by polymerase chain reaction-restriction fragment length polymorphism assay using NlaIII restriction enzyme. No statistically significant difference in either genotype distribution or allele frequency was observed between RA patients and controls. The genotype distributions and allele frequencies of rheumatoid factor negative [RF(-)] patients were similar to those of controls. However, statistical analysis of genotype revealed a significant association (chi2 = 6.42, P = 0.04) is present between RF(+) and MTNR1B SNP (rs 1562444). Although no statistically significant difference in allele frequency between RF(+) and controls was observed (chi2 = 2.75, P = 0.10), the results might suggest that MTNR1B SNP (rs 1562444) is associated with the presence of RF in RA. This is the first study, to our knowledge, to report a positive genetic relationship between melatonin and RA.

We examined the effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), the dephosphorylated form of AICA ribotide (also termed "ZMP"), an intermediate of purine biosynthesis, on interleukin (IL)-2 production in T cells. AICAR inhibited IL-2 production in Jurkat T cells and peripheral blood lymphocytes activated with PMA plus ionomycin (PMA/Io) or with monoclonal anti-CD3 plus anti-CD28. Pretreatment with 5'-iodotubercidin, an adenosine kinase inhibitor, enhanced AICAR suppression of IL-2 production, suggesting that AICAR, not ZMP, is responsible for IL-2 suppression. We then showed that AICAR inhibited PMA/Io-induced IL-2 mRNA expression and IL-2 promoter activation. AICAR inhibited DNA binding and transcriptional activation of NF-AT and to a lesser extent AP-1, but not NF-kappaB, in PMA/Io-activated Jurkat cells. Finally, we found that AICAR inhibited PMA/Io-induced phosphorylation of GSK-3 but not phosphorylation of ERK1/2, p38, and JNK. These results suggest that AICAR exerts its immunosuppressive effect in activated Jurkat cells by inhibiting GSK-3 phosphorylation and NF-AT activation.