• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.790-798
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• 2016

Chlamydiae, obligate intracellular bacteria, are associated with a variety of human diseases. The chlamydial life cycle undergoes a biphasic development: replicative reticulate bodies (RBs) phase and infectious elementary bodies (EBs) phase. At the end of the chlamydial intracellular life cycle, EBs have to be released to the surrounded cells. Therefore, the interactions between Chlamydiae and cell death pathways could greatly influence the outcomes of Chlamydia infection. However, the underlying molecular mechanisms remain elusive. Here, we investigated host cell death after Chlamydia infection in vitro, in L929 cells, and showed that Chlamydia infection induces cell necrosis, as detected by the propidium iodide (PI)-Annexin V double-staining flow-cytometric assay and Lactate dehydrogenase (LDH) release assay. The production of reactive oxygen species (ROS), an important factor in induction of necrosis, was increased after Chlamydia infection, and inhibition of ROS with specific pharmacological inhibitors, diphenylene iodonium (DPI) or butylated hydroxyanisole (BHA), led to significant suppression of necrosis. Interestingly, live-cell imaging revealed that Chlamydia infection induced lysosome membrane permeabilization (LMP). When an inhibitor upstream of LMP, CA-074-Me, was added to cells, the production of ROS was reduced with concomitant inhibition of necrosis. Taken together, our results indicate that Chlamydia infection elicits the production of ROS, which is dependent on LMP at least partially, followed by induction of host-cell necrosis. To our best knowledge, this is the first live-cell-imaging observation of LMP post Chlamydia infection and report on the link of LMP to ROS to necrosis during Chlamydia infection.

• Journal of Life Science
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• v.31 no.5
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• pp.527-535
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• 2021

Lysosomes are organelles surrounded by membranes that contain acid hydrolases; they degrade proteins, macromolecules, and lipids. According to nutrient conditions, lysosomes act as signaling hubs that regulate intracellular signaling pathways and are involved in the homeostasis of cells. Therefore, the lysosomal dysfunction occurs in various diseases, such as lysosomal storage disease, neurodegenerative diseases, and cancers. Multiple forms of stress can increase lysosomal membrane permeabilization (LMP), resulting in the induction of lysosome-mediated cell death through the release of lysosomal enzymes, including cathepsin, into the cytosol. Here we review the molecular mechanisms of LMP-mediated cell death and the enhancement of sensitivity to anticancer drugs. Induction of partial LMP increases apoptosis by releasing some cathepsins, whereas massive LMP and rupture induce non-apoptotic cell death through release of many cathepsins and generation of ROS and iron. Cancer cells have many drug-accumulating lysosomes that are more resistant to lysosome-sequestered drugs, suggesting a model of drug-induced lysosome-mediated chemoresistance. Lysosomal sequestration of hydrophobic weak base anticancer drugs can have a significant impact on their subcellular distribution. Lysosome membrane damage by LMP can overcome resistance to anticancer drugs by freeing captured hydrophobic weak base drugs from lysosomes. Therefore, LMP inducers or lysosomotropic agents can regulate lysosomal integrity and are novel strategies for cancer therapy.

• Journal of Life Science
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• v.27 no.10
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• pp.1145-1151
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• 2017

In this study, we report on a simple, efficient method for obtaining penicillin G amidase (PGA) from recombinant Escherichia coli using a formulation mixed with detergent and lysozyme. Research was conducted on the extraction efficiency of PGA from the periplasmic space in cells in terms of the type of detergent, detergent concentration, pH, reaction time, and temperature of permeabilization. The extraction yield of PGA in the formulated surfactant/lysozyme treatment was increased by approximately (55-65 U/ml) in comparison with that in the single surfactant treatment. The released PGA solution was concentrated and exchanged with buffer using an ultrafiltration (U/F) system. The yields of diatomite filtration, membrane filtration (M/F), and U/F were 69.7%, 93.8%, and 77.3%, respectively. A total of 212 KU of PGA was recovered. At the 25-L culture scale, the overall yield of extraction using the mixed surfactant/lysozyme method was 49.2%. The specific activity of extracted PGA was 11 U/mg in protein. The concentrated PGA solution was immobilized on microporous silica beads without further purification of PGA. The total immobilization yield of PGA on the resin was 48.7%, while the enzyme activity was 101 U/g. The immobilized PGA was successfully used to produce 6-APA from penicillin G. Our results indicated that a simple extraction method from periplasmic space in E. coli may be used for the commercial scale production of ${\beta}-lactam$ antibiotics using immobilized PGA.

• KSBB Journal
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• v.20 no.2 s.91
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• pp.100-105
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• 2005

Removal of nitrogen compound from waste water is essential and often accomplished by biological process. Denitrification bacterium, Paracoccus denitrificans (KCTC 2350) is employed to estimate the denitrification ability and the characteristics. In the immobilized biological reactor system, the measurement of absolute amount of active strain in the reactor is comparatively difficult or impossible. In this. study, a reactor was designed with the unwoven texture wrapped peep holed plastic tube to calculate the absolute amount of active strain by comparing the activity of the permeabilized and or immobilized reactor and the free cell reactor The reactor system was continuous stirred tank reactor and the reaction rate of substrate consumption was assumed to satisfy the Michaelis-Menten equation. The effluent concentration of nitrate and nitrite was measured to estimate the apparent parameter of Michaelis-Menten equation. As a result, we found that the amount of immobilized active strain was figured out to be half of the total active strain in the reactor and the time required to be reached in the equilibrium state in the permeabilized and or immobilized reactor system was figured out to be shorter than that of the free cell reactor system.

• KSBB Journal
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• v.6 no.3
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• pp.249-254
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• 1991

Permeabilization of Zymomonas mobilis with CTAB(Cetyltrimethylammoniumbromide) was investigated in order to obtain stable process for sorbitol production in the immobilized system. The optimum conditions for sorbitol formation were obtained in the case of using cells treated with 0.2% CTAB at$ 4^{\circ}C$ for 10 min. Permeabilized cells were treated with glutaraldehyde to cross-link the internal enzyme for the improvement of the enzyme stability. In this way, no significant loss of enzyme activity was apparent during 30-day operation in a continuous process. The productivity of the continuous process at dilution rate 0.2h-1 was 6.51g/1/h for sorbitol. The CTAB permeabilized cells could be used to produce sorbitol in the long term continuous process.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.343-348
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• 1994

Enterobacter sp. producing -$\beta$-galactosidase with high transgalactosylation activity was isolated from dairy wastewater. The isolate had common biochemical features to E. aerogenes and E. cloacae. Enzyme production increased as the cell mass increased with optimum enzyme activity of 0.21 Unit/mg-protein (o-nitro-phenyl-$\beta$ -D-galactoside (ONPG) as substrate) until 8 hr of culture. Whole cells permeabilized by toluene were used to produce galacto-oligosaccharide. Optimum toluene concentration, temperature and pH for -$\beta$-galactosidase activity of permeabilized whole cells were 10% (v/v), $50^{\circ}C$ and 6.0, respectively. A maximum of 38% (w/w) of galacto-oligosaccharide was obtained with lactose concentration of 20% (w/w) at $40^\{\circ}C$ and pH 6.0.

• Mycobiology
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• v.43 no.1
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• pp.1-8
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• 2015

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

• Bulletin of the Korean Chemical Society
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• v.35 no.2
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• pp.419-424
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• 2014

The pro-apoptotic BCL-2 family protein BID activates BAK and/or BAX, which form oligomeric pores in the mitochondrial outer membrane. This results in the release of cytochrome c into the cytoplasm, initiating the apoptotic cascade. Here, we utilized liposomes encapsulating sulfo-rhodamine at a controlled temperature to improve upon a previously reported assay system with enhanced sensitivity and specificity for measuring membrane permeabilization by BID-dependent BAK activation. BAK activation was inhibited by BCL-$X_L$ protein but not by a mutant protein with impaired anti-apoptotic activity. With the assay system, we screened a chemical library and identified several compounds including trifluoperazine, a mitochondrial apoptosis-induced channel blocker. It inhibited BAK activation by direct binding to BAK and blocking the oligomerization of BAK.

• Journal of Integrative Natural Science
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• v.16 no.3
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• pp.97-102
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• 2023

The gasdermins are a family of recently identified pore-forming effector proteins that cause membrane permeabilization and pyroptosis, a lytic pro-inflammatory type of cell death. A role in the regulation of cell proliferation and/or differentiation is suggested by the differentiation status-specific expression of gasdermin proteins in epithelial tissues. One of the GSDM protein is Gasdermin A (GSDMA), which decreased in stomach and esophageal cancers, suggesting a tumor suppressor role. GSDMA receptor antagonists have been researched as potential treatments for inflammatory diseases and baldness. GSDMA's significance in a wide range of disorders makes it an important therapeutic target. As a result, homology modelling of the GSDMA receptor was undertaken in the current study using the crystal structures of Mus musculus (GSDMA3), Human gasdermin D (GSDMD), and Murine gasdermin D (murine GSDMD). The best model was chosen based on the validation results after 20 models were developed utilising single template-based approaches. The generated structures can be used for further binding site and docking studies in the future.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.1
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• pp.51-57
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• 2010

It was hypothesized that NaF induces calcium sensitization in $Ca^{2+}$-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with $\beta$-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to $Ca^{2+}$ (decreased $EC_{50}$ and increased $E_{max}$). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in $Ca^{2+}$-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and $GTP{\gamma}S$-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a $Ca^{2+}$ dependent manner in $\beta$-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.