Ji Hyung Chung

Peripheral Cytokines and Chemokines in Alzheimer’s Disease

A chronic inflammatory process has been implicated in the neuropathology of Alzheimer’s disease (AD). The present review focuses on the current knowledge of circulating serum and plasma biomarkers of AD that are linked to inflammatory reactions. There is abundant evidence that inflammatory mechanisms within the central nervous system contribute to cognitive impairment via cytokine-mediated interactions between neurons and glial cells. Interleukins 1, 4, 6, 10, 12, 16, and 18, tumour necrosis factor, and several chemokines have been suggested as biomarkers of AD. Nonetheless, data on circulating cytokine levels are somewhat inconsistent with regard to peripheral cytokine dysregulation in AD. In summary, definite statements concerning differences in inflammatory biomarkers between controls and AD patients will require the use of sensitive multiplex assays in large patient groups in conjunction with measures of disease severity.

Mesenchymal Stem Cells Pretreated with Delivered Hph‐1‐Hsp70 Protein Are Protected from Hypoxia‐Mediated Cell Death and Rescue Heart Functions from Myocardial Injury

Mesenchymal stem cell (MSC) therapy for myocardial injury has inherent limitations due to the poor viability of MSCs after cell transplantation. In this study, we directly delivered Hsp70, a protein with protective functions against stress, into MSCs, using the Hph‐1 protein transduction domain ex vivo for high transfection efficiency and low cytotoxicity. Compared to control MSCs in in vitro hypoxic conditions, MSCs delivered with Hph‐1‐Hsp70 (Hph‐1‐Hsp70‐MSCs) displayed higher viability and anti‐apoptotic properties, including Bcl2 increase, reduction of Bax, JNK phosphorylation and caspase‐3 activity. Hsp70 delivery also attenuated cellular ATP‐depleting stress. Eight animals per group were used for in vivo experiments after occlusion of the left coronary artery. Transplantation of Hph‐1‐Hsp70‐MSCs led to a decrease in the fibrotic heart area, and significantly reduced the apoptotic positive index by 19.5 ± 2%, compared to no‐treatment controls. Hph‐1‐Hsp70‐MSCs were well‐integrated into the infarcted host myocardium. The mean microvessel count per field in the infarcted myocardium of the Hph‐1‐Hsp70‐MSC‐treated group (122.1 ± 13.5) increased relative to the MSC‐treated group (75.9 ± 10.4). By echocardiography, transplantation of Hph‐1‐Hsp70‐MSCs resulted in additional increases in heart function, compared to the MSCs‐transplanted group. Our results may help formulate better clinical strategies for in vivo MSC cell therapy for myocardial damage. STEM CELLS 2009;27:2283–2292

1H nuclear magnetic resonance based metabolic urinary profiling of patients with ischemic heart failure

OBJECTIVES We sought to identify metabolic pathways characterizing human heart failure (HF) using ¹NMR based urinary metabolomic analysis in conjunction with multivariate statistics. DESIGN AND METHODS Patients with systolic HF of ischemic origin (n=15) and healthy controls (n=20) participated in this study. Patients with type 2 diabetes mellitus were excluded. RESULTS The results showed that the urine of the HF patients had higher levels of metabolites for acetate (p<0.05) and acetone (p<0.01) compared to the healthy controls. In addition, there was a perturbation in methylmalonate metabolism as shown by increased urinary levels of methylmalonic acid (p<0.001) in the HF patients. HF patients also had increased urinary levels of cytosine (p<0.01) and phenylacetylglycine (p<0.01) and decreased 1-methylnicotinamide (p<0.05) compared to healthy controls. CONCLUSIONS TCA cycle metabolites and fatty acid metabolism were modified in the HF patients, indicating altered energy metabolism. Moreover, perturbations of metabolism in nucleotide and methylmalonate were observed.

Ubiquitin ligase CHIP induces TRAF2 proteasomal degradation and NF‐κB inactivation to regulate breast cancer cell invasion

Transcriptional factor nuclear factor‐kappaB (NF‐κB) plays a crucial role in human breast cancer cell invasion and metastasis. The carboxyl terminus of Hsc70‐interacting protein (CHIP) is a U‐box‐type ubiquitin ligase that induces ubiquitination and proteasomal degradation of its substrate proteins. In this study, we investigated the role of CHIP in the NF‐κB pathway in the invasion of MDA‐MB‐231 cells, a highly aggressive breast cancer cell line. We showed that overexpression of CHIP significantly inhibits the invasion of the MDA‐MB‐231 cells. The overexpression of CHIP suppressed expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase‐9 (MMP‐9) in MDA‐MB‐231 cells. Moreover, CHIP strongly inhibited the nuclear localization and the transcriptional activity of NF‐κB. The activation of the IkappaB kinase complex (IKK) was also blocked by CHIP overexpression. Importantly, CHIP overexpression resulted in a significant decrease in the level of TNF receptor‐associated factor 2 (TRAF2), an upstream key player in the NF‐κB pathway. However, the level of TRAF2 was restored after treatment with a proteasome inhibitor, MG‐132. Moreover, CHIP overexpression promoted the ubiquitination of TRAF2. We also found cell invasion significantly decreased in cells transfected with TRAF2 small interfering RNA (siRNA). In contrast, when CHIP expression was suppressed by siRNA in poorly invasive MCF‐7 cells, cell invasion significantly increased in conjunction with enhanced NF‐κB activation and TRAF2 levels. Taken together, these results suggest that CHIP regulates NF‐κB‐mediated cell invasion via the down‐regulation of TRAF2. J. Cell. Biochem. 112: 3612–3620, 2011.

Quercetin up-regulates expressions of peroxisome proliferator-activated receptor γ, liver X receptor α, and ATP binding cassette transporter A1 genes and increases cholesterol efflux in human macrophage cell line

Cholesterol-laden macrophages trigger accumulation of foam cells and increase the risk of developing atherosclerosis. We hypothesized that quercetin could lower the content of cholesterol in macrophages by regulating the expression of the ATP binding cassette transporter A1 (ABCA1) gene in differentiated human acute monocyte leukemia cell line (THP-1) cells and thereby reducing the chance of forming foam cells. Quercetin, in concentrations up to 30 μM, was not cytotoxic to differentiated THP-1 cells. Quercetin up-regulated both ABCA1 messenger RNA and protein expression in differentiated THP-1 cells, and its maximum effects were demonstrated at 0.3 μM for 4 to 8 hours in incubation. In addition, quercetin increased protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptor α (LXRα) within 2 hours of treatment. Because PPARγ and LXRα are important transcriptional factors for ABCA1, quercetin-induced up-regulation of ABCA1 may be mediated by increased expression levels of the PPARγ and LXRα genes. Furthermore, quercetin-enhanced cholesterol efflux from differentiated THP-1 cells to both high-density lipoprotein (HDL) and apolipoprotein A1. Quercetin at the dose of 0.15 μM elevated the cholesterol efflux only for HDL. At the dose of 0.3 μM, quercetin demonstrated effects both on HDL and apolipoprotein A1. Our data demonstrated that quercetin increased the expressions of PPARγ, LXRα, and ABCA1 genes and cholesterol efflux from THP-1 macrophages. Quercetin-induced expression of PPARγ and LXRα might subsequently affect up-regulation of their target gene ABCA1. Taken together, ingestion of quercetin or quercetin-rich foods could be an effective way to improve cholesterol efflux from macrophages, which would contribute to lowering the risk of atherosclerosis.

Bioplex analysis of plasma cytokines in Alzheimer’s disease and mild cognitive impairment

Inflammatory mechanisms within the central nervous system contribute to cognitive impairment via cytokine-mediated interactions between neurons and glial cells. Sixty-nine subjects were consecutively recruited from October 2005 to February 2007. Fifteen individuals were excluded from the study and we ultimately enrolled 19 not cognitively impaired subjects, 25 mild cognitive impairment patients, and 10 Alzheimers disease patients. To examine the inflammatory markers of mild cognitive impairment and Alzheimers disease, we measured the plasma concentrations of 23 cytokines using a bioplex assay. The results showed that the macrophage migration inhibitory factor was higher in mild cognitive impairment and in Alzheimers disease patients compared with the not cognitively impaired group; the results also showed that monokine induced by gamma interferon was higher in Alzheimers disease patients than in not cognitively impaired subjects, as well as those of the mild cognitive impairment group [corrected].

Repair activities of human 8-oxoguanine DNA glycosylase are stimulated by the interaction with human checkpoint sensor Rad9-Rad1-Hus1 complex

Rad9-Rad1-Hus1 (9-1-1) is a checkpoint protein complex playing roles in DNA damage sensing, cell cycle arrest, DNA repair or apoptosis. Human 8-oxoguanine DNA glycosylase (hOGG1) is the major DNA glycosylase responsible for repairing a specific aberrantly oxidized nucleotide, 7,8-dihydro-8-oxoguanine (8-oxoG). In this study, we identified a novel interaction between hOGG1 and human 9-1-1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9-1-1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9-1-1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9-1-1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showed that hRad9, hRad1, and hHus1 enhanced the 8-oxoG excision and beta-elimination activities of hOGG1. In addition, the presence of hRad9, hRad1, and hHus1 enhanced the formation of covalently cross-linked hOGG1-8-oxoG/C duplex complexes, as determined by a trapping assay using NaBH(4). A trimeric human 9-1-1 complex was purified from Escherichia coli cell transformed with hRad9, His-fused hRad1, or His-fused hHus1 expressing vectors. It also showed the similar activity to enhance in vitro hOGG1 glycosylase activity, compared with individual human 9-1-1 subunits. Detection of 8-oxoG in HEK293 cells using flow cytometric and spectrofluorometric analysis revealed that over-expression of hOGG1 or human 9-1-1 reduced the formation of 8-oxoG residues following the H(2)O(2) treatment. The highest 8-oxoG reduction was observed in HEK293 cells over-expressing hOGG1 and all the three subunits of human 9-1-1. These indicate that individual human 9-1-1 subunits and human 9-1-1 complex showed almost the same abilities to enhance the in vitro 8-oxoG excision activity of hOGG1, but that the greatest effect to remove 8-oxoG residues in H(2)O(2)-treated cells was derived from the 9-1-1 complex as a whole.

Quercetin Up‐regulates LDL Receptor Expression in HepG2 Cells

Quercetin, an abundant flavonol found in fruits and vegetable, has been implicated in lowering the risk of cardiovascular disease that is often associated with high plasma levels of low density lipoprotein (LDL) cholesterol. Here we investigated whether quercetin could modulate the expression of LDL receptors (LDLR) in HepG2 cells and the possible underlying mechanisms to exert quercetins effects. We found that quercetin was able to induce LDLR expression with at least a 75 µ m concentration, which was accompanied by an increase in nuclear sterol regulatory element binding protein 2 (SREBP2). This effect was mediated by activation of c‐jun‐N‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK) signalling pathways as implicated by experiments using chemical inhibitors of each pathway. When cells were challenged with protein synthesis inhibitors in quercetin‐activated LDLR transcription, LDL mRNA levels were not significantly affected by cycloheximide but puromycin abolished quercetin‐induced LDLR transcription. Taken together, we conclude that quercetin can initiate LDLR transcription by enhancing SREBP2 processing, but new protein synthesis might be necessary to exert a maximum effect of quercetin in the up‐regulation of the LDLR gene. Our findings demonstrate that quercetin strongly up‐regulated LDLR gene expression, which might elicit hypolipidemic effects by increasing the clearance of circulating LDL cholesterol levels from the blood. Copyright

Plasma phospholipid fatty acid composition in ischemic stroke: Importance of docosahexaenoic acid in the risk for intracranial atherosclerotic stenosis

OBJECTIVE While data on the relationship between fatty acid (FA) composition and the risk for total stroke have accumulated, the association between FA composition and the risk for intracranial atherosclerotic stenosis (ICAS) has never been studied. We compared plasma phospholipid FA composition between non-stroke control and ischemic stroke in Korean population, to discern the FA that distinguishes ICAS from total ischemic stroke patients. METHODS Non-stroke controls (n = 215) and stroke patients (no cerebral atherosclerotic stenosis, NCAS: n = 144 and ICAS: n = 104) were finally included in the analysis. Plasma phospholipid FA compositions were analyzed. RESULTS Age, coexistence of hypertension/diabetes were significantly different among the groups. Phospholipid FA compositions were significantly different between non-stroke control and ischemic stroke patients, and interestingly, between NCAS and ICAS in stroke patients. Pattern analysis showed that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the ω3-polyunsaturated FAs were important FAs in distinguishing NCAS and ICAS in strokes. Particularly, the risk of ICAS was inversely associated with levels of DHA contents in phospholipids (OR: 0.590, 95% CI: 0.350-0.993, p < 0.05), indicating that the risk may be increased at lower levels of DHA contents. CONCLUSION DHA and EPA are important FAs for distinguishing NCAS and ICAS in strokes. Additionally, the risk of ICAS was inversely associated with the levels of phospholipid DHA, which indicates that sufficient amounts of DHA in plasma or in diet may reduce the risk of ICAS.

Identification of the genes involved in 1-deoxynojirimycin synthesis in Bacillus subtilis MORI 3K-85

Abstract1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ-producing bacterium, Bacillus subtilis MORI, was isolated from the traditional Korean fermented food Chungkookjang. In the present study, we report the identification of the DNJ biosynthetic genes in B. subtilis MORI 3K-85 strain, a DNJ-overproducing derivate of the B. subtilis MORI strain generated by γ-irradiation, xhe genomic DNA library of B. subtilis MORI 3K-85 was constructed in Escherichia coli, and clones showing α-glucosidase inhibition activity were selected. After DNA sequencing and a series of subcloning, we were able to identify a putative Operon which consists of gabT1, yktc1, and gutB1 genes predicted to encode putative transaminase, phosphatase, and oxidoreductase, respectively. When a recombinant plasmid containing this Operon sequence was transformed into an E. coli strain, the resulting transformant was able to produce DNJ into the culture medium. Our results indicate that the gabT1, yktc1, and gutB1 genes are involved in the DNJ biosynthetic pathway in B. subtilis MORI, suggesting the possibility of employing these genes to establish a large-scale microbial DNJ overproduction system through genetic engineering and process optimization.