Background: Overexpression of survivin, a known inhibitor of apoptosis, is associated with tumor progression and drug resistance in numerous malignancies, including leukemias. The aim of this study was to investigate the effect of a specific survivin small interference RNA (siRNA) on proliferation and the sensitivity of HL-60 acute myeloid leukemia (AML) cells to the chemotherapeutic drug etoposide. Materials and Methods: The cells were transfected with siRNAs using Lipofectamine $^{TM}2000$ transfection reagent. Relative survivin mRNA and protein levels were measured by quantitative real-time PCR and Western blotting, respectively. Trypan blue exclusion assays were performed to monitor tumor cell proliferation after siRNA transfection. The cytotoxic effects of etoposide and survivin siRNA, alone and in combination, on leukemic cells were determined using MTT assay. Apoptosis was assessed by ELISA cell death assay. Results: Survivin siRNA markedly reduced both mRNA and protein expression levels in a time-dependent manner, leading to distinct inhibition of cell proliferation and increased spontaneous apoptosis. Surprisingly, survivin siRNA synergistically increased the cell toxic effects of etoposide. Moreover, survivin down-regulation significantly enhanced its induction of apoptosis. Conclusions: Our study suggests that down-regulation of survivin by siRNA can trigger apoptosis and overcome drug resistance of leukemia cells. Therefore, survivin siRNA may be an effective adjuvant in AML chemotherapy.
Contributions of specific foods to absolute intake and between-person variance in 13 nutrients were examined to develop a short food frequency questionnaire (FFQ) for the Korean elderly using dietary data collected by a semiquantitative FFQ with 96 food items. The contributions of specific foods to 90% absolute nutrient intake were measured by assessing their percentage to the total consumption of a nutrient by our samples(n=98). To assess the contribution of the foods to the between-person variance in intake of each specific nutrient, stepwise multiple regression was performed. Cumulative R2 served as the measure of contribution to variation in intake. The most important food source of absolute intake was rice for energy and protein. Important foods of between person variance contribution include bread for energy and pork for protein. Important foods of absolute and variance contribution of nutrient intake were similar for fat(noodles including jajangmyun and kalguksu), calcium(anchovy and milk) and vitamin A (carrot). The number of foods necessary to account for the variation in nutrient intake among older people in Korea differ considerably by nutrient: 18 - 43 and 8-26 foods were accounted for the respective 90 and 80% of absolute intake depending on nutrients. Carbohydrates and vitamin A and C had relatively a few major sources, thus may be assessed well by a small number of foods. Conversely, iron, potassium and B vitamins appear to need longer lists of foods. Fewer numbeer (6 - 12 and 4 - 9) of foods were required for the corresponding percentages of between-person variation for all nutrients. Depending on nutrients, 0.980-0.995 and 0.924-0.987 were respective correlation coefficients of nutrient intakes measured by 96 food items with those by lists of foods for 90% and 80% of absolute intake contribution. These findings suggest that an incomplete food list on the basis of its contribution to absolute and between person variation intakes may still be effective in discriminating among individuals. The results of this study may be useful in the design and use of dietary questionnaire.
This study was carried out to know the food habits of 200 persons, 89 males and 111 females, aged 60 years and over from June 13 to July 2, 1977 in Seoul. The results are summarized as follows: 1. Changes of the food preferences The preferences about foods after 60 years of age showed in lower scores of dislikes than those before and, however, were not significantly changed. Beef was the item which 75% of the subjects liked and was the highest in desire to eat, Likes for hot, salty, and sour taste except sweet were diminished but they generally liked hot or sweet food rather than sour or salty one and also warm dish rather than cold one. A majority of them wanted to have their meals cook more soft and tender. 2. Intakes of foods The average consumptions of the basic food groups per capita per day were 475.5g of males .and 431.8g of females for cereals and grains, 119.6g and 103.6g for meats add legumes, 221.7g and 216. lg for fruits and vegetables, 52.6g and 39.4g for milk and small fishes, and 5.0g and 4.7g for fats and oils. 3. Intakes of calorie and nutrients The percentage contributions of three nutrients, carbohydrate, protein, and fats to the total amounts of calories were 75.9, 12.4, and 11.7% of mates and 75.6, 12.4, and 12.0% of females. Women had a slightly higher percentage of caloric intake to RDA than men who didn't satisfy RDA. The subjects revealed an insufficient protein intake as well as calcium. Therefore, many older people have to be convinced of the importance of milk for both nutrients. The mean ratios for iron, niacin, and thiamin intake were higher than RDA and on the contrary. those for riboflavin, vitamin A, and ascorbic acid intake were below RDA. It seems necessary, for the aged to maintain good state of health, to promote their appetite, and to take regular mealtime because difficulty of teeth, illness, irregular mealtime, and loss of appetite had influence on less intakes of calorie and nutrients.
Alternaria brassicicola (Schwein.) invades Brassicaceae and causes black spot disease, significantly lowering productivity. Mitogen-activated protein kinases (MAPKs) and their upstream kinases, including MAPK kinases (MAPKKs) and MAPKK kinases (MAPKKK), comprise one of the most important signaling pathways determining the pathogenicity of diverse plant pathogens. The AbSte7 gene in the genome of A. brassicicola was predicted to be a homolog of yeast Ste7, a MAPKK; therefore, the function was characterized by generating null mutant strains with a gene replacement method. AbSte7 replacement mutants (RMs) had a slower growth rate and altered colony morphology compared with the wild-type strain. Disruption of the AbSte7 gene resulted in defects in conidiation and melanin accumulation. AbSte7 was also involved in the resistance pathways in salt and oxidative stress, working to negatively regulate salt tolerance and positively regulate oxidative stress. Pathogenicity assays revealed that AbSte7 RMs could not infect intact cabbage leaves, but only formed very small lesions in wounded leaves, whereas typical lesions appeared on both intact and wounded leaves inoculated with the wild-type strain. As the first studied MAPKK in A. brassicicola, these data strongly suggest that the AbSte7 gene is an essential element for the growth, development, and pathogenicity of A. brassicicola.
Objectives: The extent of bone formation that occurs at the interface of metallic implants and bone is determined by the number and activity of osteoblastic cells. Stress proteins may be contributing determinants of cell viability in altered environments. Hsp27 is a small Mr hsp which is known as a molecular chaperone. Methods: To better understand how heat shock protein 27 contributes to endosseous implant - associated metal ions affects on osteoblastic cell viability, the effect of chromium and titanium ions were compared to effects of cadmium ions in the ROS17/2.8 osteoblastic cell line. Results: ROS17/2.8 osteoblastic cell line demonstrated ion - specific reductions in growth; reductions were significantly greater for cadmium than for chromium or titanium. Chromium impaired growth of cultures without altering cell viability measured using the MTT assay. A stable transformed cell line expressing additional hsp27(clone "A7") was resistant to the toxic effects of titanium and partially protected from cadmium toxicity. Conclusions: A role for hsp27 in protection of osteoblastic cells from metal ion toxicity is supported by the chromium - induced elevations in hsp27 abundance and the behavior of the A7 cell line in response to metal ions in culture. Similar biochemical responses to altered cellular environments may contribute to the fate of tissues adjacent to select metallic implants.
A number of studies have indicated that Nurr1, which belongs to a novel class of orphan nuclear receptors (the NR4A family), is important for carcinogenesis. Here we investigated expression of Nurr1 protein in benign and malignant human prostate tissues and association with clinicopathologic features using immunohistochemical techniques. Moreover, we also investigated the ability of Nurr1 to influence proliferation, migration, invasion and apoptosis of human prostate cancer cells using small interfering RNA silencing. Immunohistochemical analysis revealed that the expression of Nurr1 protein was higher in prostate cancer tissues than in benign prostate tissue (P<0.001), levels being positively correlated with tumor T classification (P = 0.003), N classification (P = 0.017), M classification (P = 0.011) and the Gleason score (P = 0.020) of prostate cancer patients. In vitro, silencing of endogenous Nurr1 attenuated cell proliferation, migration and invasion, and induced apoptosis of prostate cancer cells. These results suggest that Nurr1 may be used as an indicator for prostate cancer progression and be useful for novel potential therapeutic strategies.
Kim, Chong-Ho;Park, Young-Soon;Chung, Koong-Nah;Elwood, Patrick C.
BMB Reports
/
v.35
no.4
/
pp.395-402
/
2002
Caveolae are small, flask-shaped, non-clathrin coated invaginations of the plasma membrane of many mammalian cells. Caveolae have a coat that includes caveolin. They have been implicated in numerous cellular processes, including potocytosis. Since the human folate receptor (hFR) and other glycosyl-phosphatidylinositol (GPI)-tailed proteins have been co-localized to caveolae, we studied the caveolin role in the hFR function by transfecting hFR and/or caveolin cDNA into Fischer rat thyroid epithelial (FRT) cells that normally do not express detectable levels of either protein. We isolated and characterized stable clones as follows: they express (1) high levels of caveolin alone, (2) hFR and caveolin, or (3) hFR alone. We discovered that hFR is correctly processed, sorted, and anchored by a GPI tail to the plasma membrane in FRT cells. No difference in the total folic acid binding or cell surface folic acid binding activity were found between the FRT cells that were transfected with hFR, or cells that were transfected with hFR and caveolin. The hFR that was expressed on the cell surface of clones that were transfected with hFR was also sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) release, and incorporated radiolabeled ethanolamine that supports the attachment of a GPI-tail on hFR. We conclude that the processing, sorting, and function of hFR is independent on the caveolin expression in FRT cells.
Nitric oxide (NO), synthesized from L-arginine by NO synthases, is a small, lipophilic, diffusible, highly reactive molecule with dichotomous regulatory roles in many biological events under physiological and pathological conditions. NO can promote apoptosis (pro-apoptosis) in some cells, whereas it inhibits apoptosis (anti-apoptosis) in other cells. This complexity is a consequence of the rate of NO production and the interaction with biological molecules such as metal ion, thiol, protein tyrosine, and reactive oxygen species. Long-lasting overproduction of NO acts as a pro-apoptotic modulator, activating caspase family proteases through the release of mitochondrial cytochrome c into cytosol, up-regulation of the p53 expression, and alterations in the expression of apoptosis-associated proteins, including the Bcl-2 family. However, low or physiological concentrations of NO prevent cells from apoptosis that is induced by the trophic factor withdrawal, Fas, $TNF{\alpha}$/ActD, and LPS. The anti-apoptotic mechanism is understood on the basis of gene transcription of protective proteins. These include: heat shock protein, hemeoxygenase, or cyclooxygenase-2 and direct inhibition of the apoptotic executive effectors caspase family protease by S-nitrosylation of the cysteine thiol group in their catalytic site in a cell specific way. Our current understanding of the mechanisms by which NO exerts both pro- and anti-apototic action is discussed in this review article.
Kim, Hyung Jun;Lee, Hyun Ji;Park, Sung Hwan;Jeon, You-Jin;Kim, Jin-Soo;Heu, Min Soo
Korean Journal of Fisheries and Aquatic Sciences
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v.48
no.2
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pp.178-186
/
2015
Protease inhibitors (PI) of trypsin and papain as target proteases from the roe of bastard halibut Paralichthys olivaceus were fractionated out using ammonium sulfate precipitation (A), DEAE 650M anion exchange chromatography (D), and Sephacryl S-300 gel filtration (S). The recovery percentages of the fractions with the strongest inhibitory activity for each fractionation method were 13% for the A4 fraction, 21.2% for the D3 fraction, and 21.3% for the S2 fraction, with specific inhibitory activities of the fractions toward trypsin and casein of 168, 139, and 218 U/mg, respectively, while no inhibition of papain was observed. The $IC_{50}$ for the trypsin-specific substrate $N{\alpha}$-benzoyl-$\small{L}$-arginine-p-nitroanilide (BAPNA) was 0.65, 1.55, 2.26, and 2.85 mg/mL for the A4, S2, A3, and D3 fractions, respectively. These results suggest that chromatographic fractionation methods (D and S) based on the molecular mass and charge of the protein were more effective at fractionating PI than was ammonium sulfate precipitation based on protein solubility, and that the bastard halibut roe extract acts as a serine protease inhibitor. Therefore, the PI fraction from fish roe might be useful for inhibiting proteases in foodstuffs, and could constitute an alternative food-grade inhibitor for the surimi industry.
Na, Chan Hyun;Hong, Ji Hye;Kim, Wan Sup;Shanta, Selina Rahman;Bang, Joo Yong;Park, Dongmin;Kim, Hark Kyun;Kim, Kwang Pyo
Molecules and Cells
/
v.38
no.7
/
pp.624-629
/
2015
Since the emergence of proteomics methods, many proteins specific for renal cell carcinoma (RCC) have been identified. Despite their usefulness for the specific diagnosis of RCC, such proteins do not provide spatial information on the diseased tissue. Therefore, the identification of cancer-specific proteins that include information on their specific location is needed. Recently, matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) based imaging mass spectrometry (IMS) has emerged as a new tool for the analysis of spatial distribution as well as identification of either proteins or small molecules in tissues. In this report, surgical tissue sections of papillary RCC were analyzed using MALDI-IMS. Statistical analysis revealed several discriminative cancer-specific m/z-species between normal and diseased tissues. Among these m/z-species, two particular proteins, S100A11 and ferritin light chain, which are specific for papillary RCC cancer regions, were successfully identified using LC-MS/MS following protein extraction from independent RCC samples. The expressions of S100A11 and ferritin light chain were further validated by immunohistochemistry of human tissues and tissue microarrays (TMAs) of RCC. In conclusion, MALDI-IMS followed by LC-MS/MS analysis in human tissue identified that S100A11 and ferritin light chain are differentially expressed proteins in papillary RCC cancer regions.
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