Cyclin-Dependent Kinase Inhibitor p27Kip1 Controls Growth and Cell Cycle Progression in Human Uterine Leiomyoma

The molecular mechanism of the cell-cycle machinery in uterine leiomyoma has not yet been fully elucidated. Among the various types of cell-cycle regulators, p27Kip1 (p27) is considered to be a potent tumor suppressor. To provide further molecular basis for understanding the progression of uterine leiomyoma, our objective was to evaluate the expression level of p27 in normal myometrium and uterine leiomyoma tissue and its effect on cytogenic growth. Western blot analysis, real-time polymerase chain reaction (PCR) and immunohistochemical staining revealed that p27 protein and messenger RNA were down-regulated in uterine leiomyoma tissue and cultured cells compared to normal myometerium. Full-length human p27 cDNA was transferred using a replication-deficient recombinant adenoviral vector (Ad.p27) into uterine leiomyoma cells and evaluated the effect on cell proliferation. Transfection of Ad.p27 into uterine leiomyoma cells resulted in the induction of apoptosis, reduction in viability and proliferation of uterine leiomyoma cells. Our results suggest a new paradigm that down-regulated p27 protein expression is the possible underlying mechanism for the growth of uterine leiomyoma and over-expression of p27 induces cell death. This study provides better understanding of the control exerted by p27 in regulating growth and disease progression of uterine leiomyoma

Detoxification: A Novel Function of BRCA1 in Tumor Suppression?

Our studies found that BRCA1 levels negatively correlate with DNA adducts induced by Benzo(a)pyrene (BaP). Pulse-chase experiments showed that the increase in BaP-induced DNA adducts in BRCA1 knockdown cells may not be associated with BRCA1’s function in nucleotide excision repair activity; rather, it may be associated with its function in modulating transcriptional regulation. BRCA1 knockdown in MCF-10A cells significantly attenuated the induction of CYP1A1 following BaP treatment indicating that the increase in BaP-induced adducts in BRCA1 knockdown cells is not CYP1A1 dependent. However, our study shows that BRCA1 defective cells may still be able to biotransform BaP by regulating other CYP enzymes, including CYP1B1. Knockdown of BRCA1 also severely affected the expression levels of two types of uridine diphosphate glucorunyltransferase (UGT1A1 and UGT1A9) and NRF2. Both UGTs are known as BaP-specific detoxification enzymes, and NRF2 is a master regulator of antioxidant and detoxification genes. Thus, we concluded that the increased amount of BaP-induced DNA adducts in BRCA1 knockdown cells is strongly associated with its loss of functional detoxification. Chromatin immunoprecipitation assay revealed that BRCA1 is recruited to the promoter/enhancer sequences of UGT1A1, UGT1A9, and NRF2. Regulation of UGT1A1 and UGT1A9 expression showed that the induction of DNA adducts by BaP is directly affected by their expression levels. Finally, overexpression of UGTs, NRF2, or ARNT significantly decreased the amount of BaP-induced adducts in BRCA1-deficient cells. Overall, our results suggest that BRCA1 protects cells by reducing the amount of BaP-induced DNA adducts possibly via transcriptional activation of detoxification gene expression

Construction of a hybrid vector which allows for temperature regulation of expression of cloned genes in cyanobacterium, Synechocystis 6803

A hybrid vector, pBVl, has been constructed which is capable of regulating expression of cloned genes in both Escherichia coli and in cyanobacterium Synechocystis 6803. Vector pBVl contains the powerful rightward promoter of bacteriophage lambda to ensure that cloned genes are transcibed at a high level. In addition, pBV] also contains a gene, cI857, encoding the lambda temperature sensitive repressor protein.The CAT gene coding for chloramphenicol aceyltransferase (CmActase) was cloned into pBV1 downstream of the lambda regulatory features to make plasmid pTC1. The expression of the CAT gene was quantified spectrophotometrically following addition of chloramphenicol and a shift in the growth temperature of the cell culture. The specific activity of CmActase increased from 540 to 5400 units within 30 minutes. Thus, using the newly constructed hybrid vector, pBVl, temperature regulation of gene expression in E. coli was observed.Thesis (M.S.)Department of Biolog

FRET-Based Detection of Enzymatic Reaction of Botulinum on Microfluidic Device

A microfluidic device was implemented to detect the enzymatic reaction of botulinum toxin A (BTA) using Förster resonance energy transfer (FRET). The microfluidic device comprised a main channel having two loading zones, a reaction chamber and a side channel perpendicular to the main channel. The reaction chamber defined by weir in the main channel was packed with microbeads. The movement of the peptide substrate and the BTA in the microfluidic device was controlled by electrophoresis, and the enzymatic reaction of the BTA was detected through the changes of the fluorescence intensity in the reaction chamber. As a result, it was observed that the enzymatic reaction was affected by the electric voltage applied for the movement of the BTA and the peptide and improved by packing the microbeads in the reaction chamber. The microfluidic device provides the tool to investigate the proteolysis of the substrate by the BTA

Studies on HG Type of Heterodera glycines in Korea

Thirteen soybean cyst nematode (SCN) (Heterodera glycines) populations collected in Korea were examined in their HG type by their reproductivity on 7 Plant Introduction indicators for the identification of HG type. Six HG types were identified, HG type 0, 2, 5, 2.5, 1.2.7, and 2.5.7. HG type 2.5 was the most frequent (4 samples, 30.8%), followed by HG type 2.5.7 (3 samples, 23.0%). About 76.9% of SCN populations were reproduced on PI 88788, followed by PI 209332 (61.5%), PI 548316 (‘Cloud’) (30.8%), and PI 548402 (‘Peking’) (7.7%). No population could reproduce on PI 90763, PI 437654, thus, they could be used for resistant source for developing SCN resistant soybean in Korea