• BMB Reports
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• v.53 no.5
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• pp.241-247
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• 2020

As an intracellular degradation system, autophagy is an essential and defensive cellular program required for cell survival and cellular metabolic homeostasis in response to various stresses, such as nutrient deprivation and the accumulation of damaged organelles. In general, autophagy flux consists of four steps: (1) initiation (formation of phagophore), (2) maturation and completion of autophagosome, (3) fusion of autophagosomes with lysosomes (formation of autolysosome), and (4) degradation of intravesicular components within autolysosomes. The number of genes and reagents that modulate autophagy is increasing. Investigation of their effect on autophagy flux is critical to understanding the roles of autophagy in many physiological and pathological processes. In this review, we summarize and discuss ways to analyze autophagy flux quantitatively and qualitatively with the use of imaging tools. The suggested imaging method can help estimate whether each modulator is an inhibitor or a promoter of autophagy and elucidate the mode of action of specific genes and reagents on autophagy processes.

• The Microorganisms and Industry
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• v.14 no.1
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• pp.17-20
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• 1988

A genetic analysis of the complex Bacillus subtilis transcriptional apparatus is essential to understand the function, regulation, and interaction of the transcriptase components during growth and sporulation. This approach in Escherichia coli has uncovered fundamental mechanisms regulating gene expression Cole and Nomura, 1986; Lindahl and Zengel, 1986) and an analysis of the B. subtilis transcriptase will allow comoparison of the E.coli system to another bacterium that has evolved under different selective pressures. To this end we used antibody probes to isolate the alpha, beta, and beta' core subunit genes from a .lambda.gtill expression vector library. To address the question of function ans regulation of the minor sigma factors that confer promoter specifity on the polymerase core (Losick et al., 1986), we used the same approach to isolate the gene for the 37,000 dalton sigma factor, sigma-37.

• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.115-121
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• 1992

In order to broaden the spectrum of substrate utilization of a Gram negative bacterium Zymomonas mobilis which has a great potential as an industrial ethanol producing microorganism, cloning of $\alpha$-amylase gene into Z. mobilis ZM4 was tried. The $\alpha$-amylase gene was isolated from Bacillus stearothermophilus. By Southern blot analysis, it was proven that the $\alpha$-amylase gene fragment was originated from a naturally occuring plasmid of B. stearothermophilus ATCC 31195. To place $\alpha$-amylase gene under the control of Z. mobilis promoter, two different Z. mobilis expression vectors, pZA26 and pLOI204, were used. The truncated $\alpha$-amylase gene was then introduced into these vectors. Both qualitative and quantitative activities of $\alpha$-amylase were observed in Z. mobilis cells harboring these plasmids with the $\alpha$-amylase gene inserted. Gas chromatographic analysis of ethanol showed that one of the Z. mobilis transconjugants was capable of producing 67 mM ethanol from rich medium(RM) containing 5% soluble starch as a sole carbon source.

• BMB Reports
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• v.31 no.5
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• pp.427-431
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• 1998

In the previous report (Choi et al., 1997), the angiogenin binding peptides identified from a phage-peptide library were analyzed by using the fusion proteins composed of the Escherichia coli maltose binding protein and its corresponding peptides. However, it was difficult to obtain a sufficient amount of the fusion proteins required for further analysis because of the low expression level. We now report a high level expression of the fusion protein and analysis of its anti-angiogenin activity. The use of strong T7 promoter and removal of signal sequence allowed about a 20-fold increase in the expression efficiency of the fusion protein. We were able to obtain about 10 mg of purified fusion protein from one liter of culture. The purified fusion protein showed angiogenin-specific affinity and inhibited the binding of biotinylated actin to human angiogenin at $IC_{50}$ of 0.6 mM. Its anti-angiogenin activity was also revealed by the chorioallantoic membrane assay.

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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• pp.138-143
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• 1997

We have designed and constructed a gene encoding novel high essential amino acid encoding protein(HEAAE). The resultant DNA fragment was tested for in vitro and in vivo expression and then cloned into plant expression vector pBI121, under the control of the cauliflower mosaic virus 35S promoter. Agrobacterium tumefaciens, strain LBA4404, was subsequently transformed with this new construct and Nicotiana tabacum var. Xanthi transgenic plants were obtained. DNA analysis by Southern procedure confirmed the presence of the multi-copy number of genes in the transformed plants. Analysis of RNA and protein synthesized in these transgenic plants demonstrated the stable expression of this gene.

• Asian Journal of Turfgrass Science
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• v.18 no.4
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• pp.211-219
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• 2004

Transgenic zoysiagrass (Zoysia japonica cv. Zenith) plants have been obtained by particle bombardment of embryogenic callus with the plasmid pSMABuba, which contains hygromycin resistance (hpt) and bialaphos resistance (bar) genes. Parameters on DNA delivery efficiency of the particle bombardment were partially optimized using transient expression assay of a chimeric $\beta-glucuronidase$(gusA) gene driven by the CaMV 35S promoter. Stably transfarmed zoysiagrass plants were recovered with a selection scheme using hygromycin. Transgenic zoysiagrass plants were confirmed by PCR analysis with specific primer for bar gene. Expression of the transgene in transformed zoysiagrass plants was demonstrated by Reverse transcriptase (RT)-PCR analysis. All the tested transgenic plants showed herbicide BastaR resistance at the field application rate of $0.1\%-0.3\%$.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4617-4622
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• 2014

Background: Several studies have previously focused on associations between the (GT)n repeat polymorphism of the heme oxygenase-1 (HO-1) gene promoter region and risk of cancers, but results are complex. We conducted the present meta-analysis to integrate relevant findings and evaluate the association between HO-1(GT)n repeat polymorphism and cancer susceptibility. Materials and Methods: Published literature was retrieved from the PubMed/MEDLINE, EMBASE and ISI Web of Science databases before November 2013. For all alleles and genogypes, odds ratios were pooled to assess the strength of the associations using either fixed-effects or random-effects models according to heterogeneity. Subgroup analysis was conducted according to ethnicity and histopathology. Results: A total of 10 studies involving 2,367 cases and 2,870 controls were identified. The results showed there was no association between HO-1 (GT)n repeat polymorphism and the cancer risk both at the allelic and genotypic level. However, in the stratified analysis, we observed an increased risk of squamous cell carcinoma in persons carrying the LL genotype and the LL+LS genotype as compared with those carrying the SS genotype. When the LS and SS genotypes were combined, the odds ratio for squamous cell carcinoma in LL-genotype carriers, were also significantly increased. No publication bias was observed. Conclusions: The LL genotype and L-allele carrying genotypes (LL+LS) of HO-1 (GT)n repeat polymorphism are potential genetic factors for developing squamous cell carcinoma. More large and well-designed studies are required for further validations.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.4
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• pp.291-297
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• 2013

Notch1 has been reported to be highly expressed in triple-negative and other subtypes of breast cancer. Mutant p53 (R280K) is overexpressed in MDA-MB-231 triple-negative human breast cancer cells. The present study aimed to determine whether the mutant p53 can be a potent transcriptional activator of the Notch1 in MDA-MB-231 cells, and explore the role of this mutant p53-Notch1 axis in curcumin-induced apoptosis. We found that curcumin treatment resulted in an induction of apoptosis in MDA-MB-231 cells, together with downregulation of Notch1 and its downstream target, Hes1. This reduction in Notch1 expression was determined to be due to the decreased activity of endogenous mutant p53. We confirmed the suppressive effect of curcumin on Notch1 transcription by performing a Notch1 promoter-driven reporter assay and identified a putative p53-binding site in the Notch1 promoter by EMSA and chromatin immunoprecipitation analysis. Overexpression of mutant p53 increased Notch1 promoter activity, whereas knockdown of mutant p53 by small interfering RNA suppressed Notch1 expression, leading to the induction of cellular apoptosis. Moreover, curcumin-induced apoptosis was further enhanced by the knockdown of Notch1 or mutant p53, but it was decreased by the overexpression of active Notch1. Taken together, our results demonstrate, for the first time, that Notch1 is a transcriptional target of mutant p53 in breast cancer cells and suggest that the targeting of mutant p53 and/or Notch1 may be combined with a chemotherapeutic strategy to improve the response of breast cancer cells to curcumin.

• BMB Reports
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• v.31 no.6
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• pp.578-584
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• 1998

In a previous paper (Kim et al., 1996a), the immediate 5' -flanking region and coding region of the human UDP-N -acetylglucosamine:-D-mannoside-1,4-Nacetylglucosaminyltransferase III (N-acetylglucosaminyitransferase- III; GnT-III) gene was reported, isolated and analyzed. Herein, we report on amplification of a new 5' -noncoding region of the GnT-III mRNA by single-strand ligation to single-stranded cDNA-PCR (5' -RACE PCR) using poly(A)+ RNA isolated from human fetal liver cells. A cDNA clone was obtained with 5' sequences (96 bp) that diverged seven nucleotides upstream from the ATG (+1) start codon. A concensus splice junction sequence, TCTCCCGCAG, was found immediately 5' to the position where the sequences of the cDNA diverged. The result suggested the presence of an intron in the 5' -noncoding region and that the cDNA was an incompletely reversetranscribed cDNA product derived from an mRNA containing a new noncoding exon. When mRNA expression of GnT-III in various human tissues and cancer cell lines was examined, Northern blot analysis indicated high expression levels of GnT-III in human fetal kidney and brain tissues, as well as for a number of leukemia and lymphoma cancer cell lines. Promoter activities of the 5' -flanking regions of exon 1 and the new noncoding region were measured in a human hepatoma cell line, HepG2, by luciferase assays. The 5'-flanking region of exon 1 was the most active, whilst that of exon 2 was inactive.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.2
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• pp.619-624
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• 2013

Background: Colorectal cancer (CRC) exists in a more common sporadic form and less common hereditary forms, associated with the Lynch syndrome, familial adenomatous polyposis (FAP) and other rare syndromes. Sporadic CRC is believed to arise as a result of close interaction between environmental factors, including dietary and lifestyle habits, and genetic predisposition factors. In contrast, hereditary forms such as those related to the Lynch syndrome result from inheritance of germline mutations of mismatch repair (MMR) genes. However, in certain cases, the influence of low penetrance alleles in familial colorectal cancer susceptibility is also undeniable. Aim: To investigate the genotype frequencies of MLH1 promoter polymorphism -93G>A and to determine whether it could play any role in modulating familial and sporadic CRC susceptibility risk. Methods: A case-control study comprising of 104 histopathologically confirmed CRC patients as cases (52 sporadic CRC and 52 Lynch syndrome patients) and 104 normal healthy individuals as controls was undertaken. DNA was extracted from peripheral blood and the polymorphism was genotyped employing PCR-RFLP methods. The genotypes were categorized into homozygous wild type, heterozygous and homozygous variants. The risk association between these polymorphisms and CRC susceptibility risk was calculated using binary logistic regression analysis and deriving odds ratios (ORs). Results: When risk association was investigated for all CRC patients as a single group, the heterozygous (G/A) genotype showed a significantly higher risk for CRC susceptibility with an OR of 2.273, (95%CI: 1.133-4.558 and p-value=0.021). When analyzed specifically for the 2 types of CRC, the heterozygous (G/A) genotype showed significantly higher risk for sporadic CRC susceptibility with and OR of 3.714, (95%CI: 1.416-9.740 and p-value=0.008). Despite high OR value was observed for Lynch syndrome (OR: 1.600, 95%CI: 0.715-3.581), the risk was not statistically significant (P=0.253). Conclusion: Our results suggest an influence of MLH1 promoter polymorphism -93G>A in modulating susceptibility risk in Malaysian CRC patients, especially those with sporadic disease.