• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.4773-4780
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• 2014

Background: To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. Materials and Methods: The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. Results: The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5nmol/L~50nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations ($IC_{50}$) of siRNA1384, siRNA1276 and siRNA1786 for 24hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50nmol/L siRNA transfection. Conclusions: Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.

• BMB Reports
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• v.40 no.4
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• pp.459-466
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• 2007

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7$\cdot}$Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

• Horticultural Science & Technology
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• v.29 no.6
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• pp.511-522
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• 2011

Flower color is one of the main target traits in the flower breeding. Recently, technological advances in genetic engineering have been successfully reported the flower colors, such as blue roses and blue carnations that are impossible to develop by traditional breeding. Accumulated knowledge-based approaches for flavonoid biosynthesis enabled to introduce novel and unique colors into flowers. These flower color modifications have been made through the regulation of flavonoid metabolic pathway - control of endogenous gene expression and introduction of foreign genes to produce novel and specific flavonoids - and the introduction of transcription factors that are known to regulate sets of genes being involving in the flavonoid biosynthetic pathway. More empirical regulation of the flavonoids metabolism requires the understanding for regulatory mechanism of intrinsic flavonoids depending on the flower crops and the very sophisticated control of flavonoid metabolic flow. In this review, we summarized successful examples of flower color modification. It might be useful to deduce the strategy for the creation of exquisite colors in flower plants.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2753-2758
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• 2014

Background: GC-binding factor 2 (GCF2) is a transcriptional regulator that represses transcriptional activity of the epidermal growth factor receptor (EGFR) by binding to a specific GC-rich sequence in the EGFR gene promoter. In addition to this function, GCF2 has also been identified as a tumor-associated antigen and regarded as a potentially valuable serum biomarker for early human hepatocellular carcinoma (HCC) diagnosis. GCF2 is high expressed in most HCC tissues and cell lines including HepG2. This study focused on the influence of GCF2 on cell proliferation and apoptosis in HepG2 cells. Materials and Methods: GCF2 expression at both mRNA and protein levels in HepG2 cells was detected with reverse transcription (RT) PCR and Western blotting, respectively. RNA interference (RNAi) technology was used to knock down GCF2 mRNA and protein expression. Afterwards, cell viability was analyzed with a Cell Counting Kit-8 (CCK-8), and cell apoptosis and caspase 3 activity by flow cytometry and with a Caspase 3 Activity Kit, respectively. Results: Specific down-regulation of GCF2 expression caused cell growth inhibition, and increased apoptosis and caspase 3 activity in HepG2 cells. Conclusions: These primary results suggest that GCF2 may influence cell proliferation and apoptosis in HepG2 cells, and also provides a molecular basis for further investigation into the possible mechanism at proliferation and apoptosis in HCC.

• Journal of Korean Society of Environmental Engineers
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• v.34 no.12
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• pp.855-862
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• 2012

Caenorhabditis elegans, a free-living nematode mainly found in the soil pore water, roles the critical function in trophic levels, energy flow, and decomposition in soil ecosystem. C. elegans is commonly used species to test soil toxicity. Recently, they are employed broadly as a test organism in nanotoxicology. In this study, a review of the toxicity of nanomaterials for C. elegans was presented based on SCI (E) papers. The nanotoxicity studies using C. elegans have been reported in 20 instances including the mechanism of toxicity. Most studies used K-medium, S-medium, and NGM (Nematode Growth Medium) plate as an exposure medium to test toxicity of nanoparticles. The effects observed include anti aging, phototoxicity, genotoxicity, and dermal effects on C. elegans exposed to nanoparticles. We found that the toxic mechanisms were related with various aspects such as lifespan abnormality, oxidative stress, distribution of particles on inter-organisms, and stress-related gene analysis. C. elegans has advantage to test toxicity of nanoparticles due to various cellular activities, full genome information, and easy observation of transparent body. C. elegans was considered to be a good test species to evaluate the nanotoxicity.