• Microbiology and Biotechnology Letters
• /
• v.32 no.2
• /
• pp.110-116
• /
• 2004

The over-expression in E. coli of the pHLN1-SO(＋) and pHLN2-80(－) plasmids cloned an insecticidal crystal protein (ICP) gene (crylAal type) from Bacillus thuringiensis var. kurstaki HD 1 was investigated through in part, the deletion of -80 bp promoter and an alternative change of cloning vector system. Two recombinant plasmids were constructed in an attempt to analyze the over-expression of the ICP in relations to its gene structure possessing only -14 bp ［Shine-Dalgarno (SD) sequence of -80 bp promoter］. Also, anther two recombinant plasmids similarly cloned the icp gene in a different vector system. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone, pHLRBS1-14 clone in which only the SD sequence in the inverted orientation icp gene appeared, was more evident than the pHLRBS2-14 clone in which only the -14 bp SD sequence of the right orientated icp gene was shown to exist. The pHLN2-80(－) clone produced more ICP proteins than the pHLRBS1-14 clone. In the two clones, pHLNUC1-80 right-oriented icp gene and the pHLNUC2-80 clone inverted-orientation icp gene in a new different vector, the pHLNUC2-80 produced more ICP proteins in E. coli system. These results indicate that the P/ac promoter, the inverted icp gene insertion and -80 bp promoter (-66 bp part of the icp gene promoters), were concerned with the expression of the icp gene in the recombinant plasmids. In addition, the expression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.

• Asian-Australasian Journal of Animal Sciences
• /
• v.31 no.5
• /
• pp.658-671
• /
• 2018

Objective: Nandrolone decanoate (ND) is an anabolic-androgenic steroid frequently used for clinical treatment. However, the inappropriate use of ND results in the reduction of serum testosterone level and sperm production. The suppressive effect of ND on testosterone production has not been investigated in detail. The present study was designed to examine the effect of ND on the expression of steroidogenic enzymes in the rat testis. Methods: Male Sprague Dawley rats at 50 days of age were subcutaneously administrated with either 2 or 10 mg of ND/kg body weight/week for 2 or 12 weeks. The changes of transcript and protein levels of steroidogenic enzymes in the testis were determined by real-time polymerase chain reaction and western blotting analyses, respectively. Moreover, immunohistochemical analysis was employed to determine the changes of immunostaining intensity of these enzymes. The steroidogenic enzymes investigated were steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage enzyme, $17{\alpha}-hydroxylase$, $3{\beta}-hydroxysteroid$ dehydrogenase, and cytochrome P450 aromatase. Results: The treatment of ND resulted in depletion of Leydig cells and sloughing of germ cells in the testis. The ND treatment caused significant expressional decreases of steroidogenic enzymes at transcript and protein levels, and the destructive effects of ND on the testis were more apparent with a higher dose and a longer period of the treatment. Evident reduction of immunostaining intensity present in Leydig cells was clearly detected by the ND treatment. Conclusion: The exposure to ND in young male results not only in histological changes of the testis but also in aberrant gene expression of testicular steroidogenic enzymes, consequently leading into the reduction of testosterone production in the testis and thus likely disruption of spermatogenesis.

• Molecules and Cells
• /
• v.43 no.10
• /
• pp.848-855
• /
• 2020

Cells assemble stress granules (SGs) to protect their RNAs from exposure to harmful chemical reactions induced by environmental stress. These SGs release RNAs, which resume translation once the stress is relieved. During stem cell differentiation, gene expression is altered to allow cells to adopt various functional and morphological features necessary to differentiate. This process induces stress within a cell, and cells that cannot overcome this stress die. Here, we investigated the role of SGs in the progression of stem cell differentiation. SGs aggregated during the neuronal differentiation of human bone marrow-mesenchymal stem cells, and not in cell lines that could not undergo differentiation. SGs were observed between one and three hours post-induction; RNA translation was restrained at the same time. Immediately after disassembly of SGs, the expression of the neuronal marker neurofilament-M (NF-M) gradually increased. Assembled SGs that persisted in cells were exposed to salubrinal, which inhibited the dephosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), and in eIF2α/S51D mutant cells. When eIF2α/S51A mutant cells differentiated, SGs were not assembled. In all experiments, the disruption of SGs was accompanied by delayed NF-M expression and the number of neuronally differentiated cells was decreased. Decreased differentiation was accompanied by decreased cell viability, indicating the necessity of SGs for preventing cell death during neuronal differentiation. Collectively, these results demonstrate the essential role of SGs during the neuronal differentiation of stem cells.

• Toxicological Research
• /
• v.31 no.4
• /
• pp.331-337
• /
• 2015

Synthetic pyrethroids (SPs) are the most common pesticides which are recently used for indoor pest control. The widespread use of SPs has resulted in the increased exposure to wild animals and humans. Recently, some SPs are suspected as endocrine disrupting chemicals (EDCs) and have been assessed for their potential estrogenicity by adopting various analyzing assays. In this study, we examined the estrogenic effects of lambda-cyhalothrin (LC) and cypermethrin (CP), the most commonly used pesticides in Korea, using BG-1 ovarian cancer cells expressing estrogen receptors (ERs). To evaluate the estrogenic activities of two SPs, LC and CP, we employed MTT assay and reverse-transcription polymerase chain reaction (RT-PCR) in LC or CP treated BG-1 ovarian cancer cells. In MTT assay, LC ($10^{-6}M$) and CP ($10^{-5}M$) significantly induced the growth of BG-1 cancer cells. LC or CP-induced cell growth was antagonized by addition of ICI 182,720 ($10^{-8}M$), an ER antagonist, suggesting that this effect appears to be mediated by an ER-dependent manner. Moreover, RT-PCR results showed that transcriptional level of cyclin D1, a cell cycle-regulating gene, was significantly up-regulated by LC and CP, while these effects were reversed by co-treatment of ICI 182,780. However, p21, a cyclin D-ckd-4 inhibitor gene, was not altered by LC or CP. Moreover, $ER{\alpha}$ expression was not significantly changed by LC and CP, while down-regulated by E2. Finally, in xenografted mouse model transplanted with human BG-1 ovarian cancer cells, E2 significantly increased the tumor volume compare to a negative control, but LC did not. Taken together, these results suggest that LC and CP may possess estrogenic potentials by stimulating the growth of BG-1 ovarian cancer cells via partially ER signaling pathway associated with cell cycle as did E2, but this estrogenic effect was not found in in vivo mouse model.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.6
• /
• pp.1337-1345
• /
• 2005

Vibrio vulnificus is the causative agent of foodborne diseases such as gastroenteritis and life-threatening septicemia. Microbial pathogenicity is a complex phenomenon in which expression of numerous virulence factors is frequently controlled by a common regulatory system. In the present study, a mutant exhibiting decreased cytotoxic activity toward intestinal epithelial cells was screened from a library of V. vulnificus mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, an open reading frame, fexA, a homologue of Escherichia coli areA, was identified and cloned. The nucleotide and deduced amino acid sequences of the fexA were analyzed, and the amino acid sequence of FexA from V. vulnificus was $84\%\;to\;97\%$ similar to those of AreA, an aerobic respiration control global regulator, from other Enterobacteriaceae. Functions of the FexA were assessed by the construction of an isogenic mutant, whose fexA gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of fexA resulted in a significant alteration in growth rate under aerobic as well as anaerobic conditions. When compared to the wild-type, the fexA mutant exhibited a substantial decrease in motility and cytotoxicity toward intestinal epithelial cell lines in vitro. Furthermore, the intraperitoneal $LD_{50}$ of the fexA mutant was approximately $10^{1}-10^{2}$ times higher than that of parental wild-type. Therefore, it appears that FexA is a novel global regulator controlling numerous genes and contributing to the pathogenesis as well as growth of V. vulnificus.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.4
• /
• pp.374-378
• /
• 2011

To find alternative genetic resources for D-serine dehydratase (E.C. 4.3.1.18, dsdA) mediating the deamination of D-serine into pyruvate, metagenomic libraries were screened. The chromosomal dsdA gene of a wild-type Escherichia coli W3110 strain was disrupted by inserting the tetracycline resistance gene (tet), using double-crossover, for use as a screening host. The W3110 dsdA::tet strain was not able to grow in a medium containing D-serine as a sole carbon source, whereas wild-type W3110 and the complement W3110 dsdA::tet strain containing a dsdA-expression plasmid were able to grow. After introducing metagenome libraries into the screening host, a strain containing a 40-kb DNA fragment obtained from the metagenomic souce derived from a compost was selected based on its capability to grow on the agar plate containing D-serine as a sole carbon source. For identification of the genetic resource responsible for the D-serine degrading capability, transposon-${\mu}$ was randomly inserted into the 40-kb metagenome. Two strains that had lost their D-serine degrading ability were negatively selected, and the two 6-kb contigs responsible for the D-serine degrading capability were sequenced and deposited (GenBank code: HQ829474.1 and HQ829475.1). Therefore, new alternative genetic resources for D-serine dehydratase was found from the metagenomic resource, and the corresponding ORFs are discussed.

• Korean Journal of Microbiology
• /
• v.48 no.2
• /
• pp.171-174
• /
• 2012

THOC1/Hpr1 is one subunit of THO complex that is an evolutionally conserved assembly involved in the mRNP packaging and mRNA export during transcription elongation. In fission yeast Schizosaccharomyces pombe, an ortholog (spHpr1) of THOC1/Hpr1 was identified based on sequence alignment. A deletion mutant in a diploid strain was constructed by replacing one of spHpr1-coding region with a $kan^r$ gene using one-step gene disruption method. Tetrad analysis showed that the sphpr1 is essential for growth. Over-expression of sphpr1 from strong nmt1 promoter caused no defects of growth and mRNA export. However, repression of the sphpr1 expression resulted in growth inhibition accompanied by accumulation of poly$(A)^+$ RNA in the nucleus. These results suggest that spHpr1 is involved in mRNA export from the nucleus to cytoplasm.

• Korean Journal of Microbiology
• /
• v.50 no.3
• /
• pp.249-253
• /
• 2014

THOC7/Mft1 is a member of the THO complex that is an evolutionally conserved assembly connecting transcription elongation with mRNP packaging and mRNA export. In fission yeast Schizosaccharomyces pombe, an ortholog (spThoc7) of THOC7/Mft1 was isolated by partial complementation of the lethality in synthetic lethal mutant, SLRsm1. A deletion mutant in a diploid strain was constructed by replacing one of spThoc7-coding region with an $ura4^+$ gene using one-step gene disruption method. Tetrad analysis showed that the spthoc7 is nonessential for growth. But the ${\Delta}thoc7$ null mutant showed slight defects of both growth and mRNA export. And the functional spThoc7-GFP protein is localized mainly in the nucleus. These results suggest that spThoc7 is also involved in mRNA export from the nucleus to cytoplasm.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.12
• /
• pp.1630-1636
• /
• 2010

During the fermentation process of Saccharomyces cerevisiae, yeast cells must rapidly respond to a wide variety of external stresses in order to survive the constantly changing environment, including ethanol stress. The accumulation of ethanol can severely inhibit cell growth activity and productivity. Thus, the response to changing ethanol concentrations is one of the most important stress reactions in S. cerevisiae and worthy of thorough investigation. Therefore, this study examined the relationship between ethanol tolerance in S. cerevisiae and a unique protein called alcohol sensitive RING/PHD finger 1 protein (Asr1p). A real-time PCR showed that upon exposure to 8% ethanol, the expression of Asr1 was continuously enhanced, reaching a peak 2 h after stimulation. This result was confirmed by monitoring the fluorescence levels using a strain with a green fluorescent protein tagged to the C-terminal of Asr1p. The fluorescent microscopy also revealed a change in the subcellular localization before and after stimulation. Furthermore, the disruption of the Asr1 gene resulted in hypersensitivity on the medium containing ethanol, when compared with the wild-type strain. Thus, when taken together, the present results suggest that Asr1 is involved in the response to ethanol stress in the yeast S. cerevisiae.

• Genomics & Informatics
• /
• v.12 no.4
• /
• pp.181-186
• /
• 2014

Genome-wide association studies have proven the highly polygenic architecture of complex diseases or traits; therefore, single-locus-based methods are usually unable to detect all involved loci, especially when individual loci exert small effects. Moreover, the majority of associated single-nucleotide polymorphisms resides in non-coding regions, making it difficult to understand their phenotypic contribution. In this work, we studied epistatic interactions associated with three common diseases using Korea Association Resource (KARE) data: type 2 diabetes mellitus (DM), hypertension (HT), and coronary artery disease (CAD). We showed that epistatic single-nucleotide polymorphisms (SNPs) were enriched in enhancers, as well as in DNase I footprints (the Encyclopedia of DNA Elements [ENCODE] Project Consortium 2012), which suggested that the disruption of the regulatory regions where transcription factors bind may be involved in the disease mechanism. Accordingly, to identify the genes affected by the SNPs, we employed whole-genome multiple-cell-type enhancer data which discovered using DNase I profiles and Cap Analysis Gene Expression (CAGE). Assigned genes were significantly enriched in known disease associated gene sets, which were explored based on the literature, suggesting that this approach is useful for detecting relevant affected genes. In our knowledge-based epistatic network, the three diseases share many associated genes and are also closely related with each other through many epistatic interactions. These findings elucidate the genetic basis of the close relationship between DM, HT, and CAD.