Characterization of Lysine Acetyltransferase Activity of Recombinant Human ARD1/NAA10.

Arrest defective 1 (ARD1), also known as N(alpha)-acetyltransferase 10 (NAA10) was originally identified as an N-terminal acetyltransferase (NAT) that catalyzes the acetylation of N-termini of newly synthesized peptides. After that, mammalian ARD1/NAA10 expanded its' role to lysine acetyltransferase (KAT) that post-translationally acetylates internal lysine residues of proteins. ARD1/NAA10 is the only enzyme with both NAT and KAT activities. However, recent studies on the role of human ARD1/NAA10 (hARD1/NAA10) in lysine acetylation are contradictory, as crystal structure and in vitro acetylation assay results revealed the lack of KAT activity. Thus, the role of hARD1/NAA10 in lysine acetylation is still debating. Here, we found a clue that possibly explains these complicated and controversial results on KAT activity of hARD1/NAA10. Recombinant hARD1/NAA10 exhibited KAT activity, which disappeared soon in vitro. Size-exclusion analysis revealed that most recombinant hARD1/NAA10 formed oligomers over time, resulting in the loss of KAT activity. While oligomeric recombinant hARD1/NAA10 lost its ability for lysine acetylation, its monomeric form clearly exhibited lysine acetylation activity in vitro. We also characterized the KAT activity of hARD1/NAA10 that was influenced by several experimental conditions, including concentration of reactants and reaction time. Taken together, our study proves that recombinant hARD1/NAA10 exhibits KAT activity in vitro but only under accurate conditions, including reactant concentrations and reaction duration.

Impact of calcineurin inhibitors on rat glioma cells viability.

Background: Although kidney transplantation outcomes have improved dramatically after using calcineurin inhibitors (CNIs), CNI toxicity continues to be reported and the mechanism remains uncertain. Here, we investigated the neurotoxicity of CNIs by focusing on the viability of glioma cells. Methods: Glioma cells were treated with several concentrations of CNIs for 24 hours at 37℃ and their cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: Exposure to 0, 0.25, 0.5, 2.5, 5.0, and 10.0 mM concentrations respectively showed 100%, 64.3%, 61.3%, 68.1%, 62.4%, and 68.6% cell viability for cyclosporine and 100%, 38.6%, 40.8%, 43.7%, 37.8%, and 43.0% for tacrolimus. The direct toxic effect of tacrolimus on glioma cell viability was stronger than that of cyclosporine at the same concentration. Conclusion: CNIs can cause neurological side effects by directly exerting cytotoxic effects on brain cells. Therefore, we should carefully monitor the neurologic symptoms and level of CNIs in kidney transplant patients.

Cyanate improves insulin sensitivity and hepatic steatosis in normal and high fat-fed mice: Anorexic and antioxidative effects.

Obesity is an important contributing factor to progression of chronic kidney disease. Cyanate, known as uremic toxin, is an electrophile produced spontaneously from urea or by myeloperoxidase-catalyzed oxidation of thiocyanate. Herein, we explored metabolic effects of cyanate in normal chow diet (NCD)- and high fat diet (HFD)-fed mice. Mice were treated with cyanate (1 mg/mL in drinking water) and fed NCD or HFD. Peritoneal glucose tolerance test (PGTT) and insulin tolerance test (ITT) were performed. Blood urea nitrogen (BUN) and creatinine concentrations were determined. Kidney and liver tissues were analyzed for reactive oxygen species (ROS) and lipid accumulations. Human albumin was carbamylated and evaluated for ROS scavenging activities. Contrary to our expectations, we found that cyanate treatment improved increased insulin sensitivity and alleviated hepatic steatosis in NCD- and HFD-fed mice. PGTT and ITT revealed faster and immediate glucose clearance in cyanate-treated NCD- and HFD-fed mice. Histological analysis of kidney and serum levels of BUN and creatinine showed no significant differences between cyanate-treated and control mice groups. Cyanate treatment reduced appetite and body weight in both NCD- and HFD-fed mice groups. Cyanate also decreased lipid peroxidation levels in the sera and the kidney, attenuated ROS levels in the kidney, which lead us to the findings that cAlb significantly reduced ROS levels compared to Alb in Caki-1 kidney and human umbilical vein endothelial cells. The results in this study may indicate that cyanate improves insulin sensitivity and hepatic steatosis possibly via exerting anorexic and antioxidative effects.

Verification of learner's differences by team-based learning in biochemistry classes.

PURPOSE: We tested the effect of team-based learning (TBL) on medical education through the second-year premedical students' TBL scores in biochemistry classes over 5 years. METHODS: We analyzed the results based on test scores before and after the students' debate. The groups of students for statistical analysis were divided as follows: group 1 comprised the top-ranked students, group 3 comprised the low-ranked students, and group 2 comprised the medium-ranked students. Therefore, group T comprised 382 students (the total number of students in group 1, 2, and 3). To calibrate the difficulty of the test, original scores were converted into standardized scores. We determined the differences of the tests using Student t-test, and the relationship between scores before, and after the TBL using linear regression tests. RESULTS: Although there was a decrease in the lowest score, group T and 3 showed a significant increase in both original and standardized scores; there was also an increase in the standardized score of group 3. There was a positive correlation between the pre- and the post-debate scores in group T, and 2. And the beta values of the pre-debate scores and "the changes between the pre- and post-debate scores" were statistically significant in both original and standardized scores. CONCLUSION: TBL is one of the educational methods for helping students improve their grades, particularly those of low-ranked students.

[Antioxidant status in nonalcoholic steatohepatitis].

BACKGROUND/AIMS: Nonalcoholic steatohepatitis (NASH) is chronic liver disease that can potentially progress to end stage liver disease. Oxidative stress to the vulnerable fatty liver has been reported as a key mechanism in development of NASH. Several antioxidant pathways have been identified, but reports that involved quantitative analysis of each antioxidant systems are rare, and these reports have shown various results. So, we investigated antioxidant status and the degree of oxidative stress by measuring several antioxidant enzymes, the total antioxidant status (TAS), and the metabolites of superoxide in NASH patients. METHODS: Nineteen NASH patients who were confirmed by liver biopsy and fifteen controls were involved in this study. The levels of body mass index (BMI), AST, ALT, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, TAS, hydrogen peroxide (H2O2), and malondialdehyde (MDA) were compared between both groups. The relationship between the histologic severity and the levels of each antioxidants were analyzed in the NASH group. RESULTS: The activities of SOD and catalase were lower in the NASH group. The concentrations of TAS and H2O2 were higher in NASH group. The level of GPx and MDA showed no significant differences between both groups. There were no significant relationships between the above variables and the pathological severity. CONCLUSIONS: The disturbed metabolism of superoxide due to the decreased activities of SOD and catalase seem to be important in the pathogenesis of NASH. Further investigations about the nonenzymatic secondary antioxidant mechanism are necessary because the TAS was higher for the NASH group. The lack of difference between both groups for the concentration of MDA indicates that mechanisms other than lipid peroxidation also may be important in the pathogenesis of NASH.

Carbamylated low-density lipoprotein attenuates glucose uptake via a nitric oxide-mediated pathway in rat L6 skeletal muscle cells.

Carbamylation is a cyanate-mediated posttranslational modification. We previously reported that carbamylated low-density lipoprotein (cLDL) increases reactive oxygen species and apoptosis via a lectin-like oxidized LDL receptor mediated pathway in human umbilical vein endothelial cells. A recent study reported an association between cLDL and type 2 diabetes mellitus (T2DM). In the current study, the effects of cLDL on glucose transport were explored in skeletal muscle cells. The effect of cLDL on glucose uptake, glucose transporter 4 (GLUT4) translocation, and signaling pathway were examined in cultured rat L6 muscle cells using 2-deoxyglucose uptake, immunofluorescence staining and western blot analysis. The quantity of nitric oxide (NO) was evaluated by the Griess reaction. The effect of native LDL (nLDL) from patients with chronic renal failure (CRF-nLDL) on glucose uptake was also determined. It was observed that cLDL significantly attenuated glucose uptake and GLUT4 translocation to the membrane, which was mediated via the increase in inducible nitric oxide synthase (iNOS)-induced NO production. Tyrosine nitration of the insulin receptor substrate-1 (IRS­1) was increased. It was demonstrated that CRF-nLDL markedly reduced glucose uptake compared with nLDL from healthy subjects. Collectively, these findings indicate that cLDL, alone, attenuates glucose uptake via NO-mediated tyrosine nitration of IRS­1 in L6 rat muscle cells and suggests the possibility that cLDL is involved in the pathogenesis of T2DM.

Mutations of the BAK gene are infrequent in advanced gastric adenocarcinomas in Koreans.

The bcl-2 homologue antagonist/killer (BAK) is a potently apoptosis-inducing gene and plays an important role in modulating apoptosis in epithelial cells. We have analyzed the mutation of the entire coding region of BAK gene in 107 Korean advanced gastric adenocarcinomas by polymerase chain reaction-single strand conformation polymorphism and sequencing. Homozygous deletions were not found in these samples. Only three cases of 107 gastric adenocarcinomas (2.8%) exhibited the BAK mutations. Two of them exhibited missense mutations and the remaining one had a silent mutation. All of these mutations were exclusively detected in exon 2. Mutations in the BAK gene were observed only in advanced gastric adenocarcinomas with extensive metastases of regional lymph nodes. The data presented here suggest that the mutations of BAK gene rarely occurred in advanced gastric adenocarcinomas.

Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells.

Tetrandrine, which is isolated from Chinese herb Stephania tetrandrae, possesses anti-inflammatory, immunosuppressive, and cytoprotective properties. Though it was previously shown that tetrandrine causes a G1 blockade and apoptosis in various cell types, however, the mechanism by which tetrandrine initiates apoptosis remains poorly understood. In present study, we investigated the mechanisms of apoptosis induced by tetrandrine in U937 leukemia cells. Tetrandrine inhibited U937 cell growth by inducing apoptosis. After treatment of U937 cells with tetrandrine (10microM) for 24h, alteration of cell morphology, chromatin fragmentation, cytochrome c release, and caspase activation were observed. Tetrandrine also induced early oxidative stress, which resulted in activation of JNK, but not ERK and p38 MAPK. A broad-spectrum caspase inhibitor and antioxidants significantly blocked tetrandrine-induced caspase-3 activation. However, inhibition of the JNK activity with SP600125 did not block tetrandrine-induced apoptosis. Tetrandrine-induced apoptosis of U937 cells also required activity of PKC-delta, because pretreatment with a specific PKC-delta inhibitor greatly blocked tetrandrine-induced caspase-3 activation. In addition, the apoptotic response to tetrandrine was significantly attenuated in dominant-negative PKC-delta transfected MCF-7 cells, suggesting that PKC-delta plays an important role in tetrandrine-induced apoptosis and can induce caspase activation. These results suggest that tetrandrine induces oxidative stress, JNK activation, and caspase activation. However, JNK activation by ROS is not involved in the tetrandrine-induced apoptosis. In addition, tetrandrine induces caspase-dependent generation of a catalytically active fragment of PKC-delta, and this fragment also appears to play a role in the activation of caspases.

Arsenic trioxide-induced apoptosis in U937 cells involve generation of reactive oxygen species and inhibition of Akt.

Arsenic trioxide has recently been shown to inhibit growth and induce apoptosis in acute promyelocytic leukemia (APL), but little is known about the molecular mechanisms mediating these effects. In the present study, we determined the molecular pathways that lead to apoptosis after treatment of cells with arsenic trioxide. Arsenic trioxide treatment of U937 cells leads to apoptosis, which is accompanied by activation of caspase 3 (as measured by decreased levels of the 32 kDa inactive form and increased proteolytic cleavage of PLC-gamma1). The broad-range caspase inhibitor z-VAD-fmk inhibits this induction of apoptosis, supporting a direct link between caspase activation and arsenic trioxide induction of apoptosis. This activation of apoptosis is accompanied by release of cytochrome c, down-regulation of cIAP1, and inactivation of Akt. Bcl-2 overexpression attenuates arsenic trioxide-induced apoptosis in U937 cells by inhibition of caspase 3 activity, but not inhibition of Akt. In addition, arsenic trioxide-induced apoptosis was caused by the generation of reactive oxygen species, which was prevented by antioxidant NAC (N-acetyl-cysteine). Co-treatment with NAC markedly prevented dephosphorylation of Akt, activation of caspase 3, and down-regulation of cIAP1. These data indicate that arsenic trioxide can cause cell damage by inactivating the Akt-related cell survival pathway and generating the reactive oxygen species, providing a new mechanism for arsenic trioxide-induced apoptosis.

Ciglitazone enhances ovarian cancer cell death via inhibition of glucose transporter-1.

Ciglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist and improves insulin sensitivity. Apart from antidiabetic activity, ciglitazone elicits inhibitory effects on cancer cell growth. Recent studies indicate that glucose metabolism plays a key role in malignant diseases. Significant increase in glucose consumption is found under malignant conditions. The role of ciglitazone in cancer cell death in relation to glucose metabolism is unclear. Thus we designed this study to determine the effect of ciglitazone on glucose metabolism. First, we found ciglitazone inhibited glucose uptake in ovarian cancer cells but did not affect hexokinase activity. Ciglitazone decreased expression levels of glucose transporter-1 (GLUT-1). We also found that ciglitazone and siGLUT-1 treatments induced cell death in ovarian cancer cells. We identified that ciglitazone decreased expressions of specific protein 1 (Sp-1) and ß-catenin while increased phosphorylation levels of AMP-activated protein kinase. In vivo study using NOD-scid IL2Rgamma(null) mice confirmed that ciglitazone significantly decreased ovarian cancer mass transplanted onto the back of the mice. Finally, we determined GLUT-1 expressions in patients with serous type ovarian cancer and found that GLUT-1 expression was markedly increased in cancer patients and expression level was proportional to the degree of cancer stages. These results suggest that ciglitazone induces apoptosis in ovarian cancer cells by the inhibition of GLUT-1 and provides a possible therapeutic effect of ciglitazone as an adjuvant drug in the treatment of ovarian cancer.