• Microbiology and Biotechnology Letters
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• v.22 no.2
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• pp.143-151
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• 1994

In the course of the study on strain inprovement by protoplast fusion, Lactobacillus acidophilus 88 protoplasts production and regeneration conditions were investigated. This strain produced a bacteriocin that revealed strong inhibitory activity against various indicator strains, especially L. helveticus CNRZ 1096. Protoplasts of L. acidophilus 88 strains were very efficiently obtained by treatment with 125 $\mu $g/ml lysozyme in a protoplast forming buffer containing 20 mM N-2 hydroxy-ethtl-piperazine-N'-2-ethane-sulfonic acid(HEPES, pH 7.0) and 1M sucrose at 37$\circ $C for 30 min. Hovever, treatment with mutanolysin was not effective for the production of L. acidophilus 88 protoplasts under the same conditions. High protoplast yield was obtained form the cells at the middle to late logarithmic growth phase in the de Man, Rogosa and Sharpe(MRS) medium. Regeneration was efficiently accomplished with the MRS medium containing 10% sucrose.

• Molecules and Cells
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• v.42 no.12
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• pp.893-905
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• 2019

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion processes that closely related to their function. Disruption of mitochondrial dynamics has been demonstrated in acute kidney injury (AKI), which could eventually result in cell injury and death. Previously, we reported that augmenter of liver regeneration (ALR) alleviates renal tubular epithelial cell injury. Here, we gained further insights into whether the renoprotective roles of ALR are associated with mitochondrial dynamics. Changes in mitochondrial dynamics were examined in experimental models of renal ischemia-reperfusion (IR). In a model of hypoxia-reoxygenation (HR) injury in vitro, dynamin-related protein 1 (Drp1) and mitochondrial fission process protein 1 (MTFP1), two key proteins of mitochondrial fission, were downregulated in the Lv-ALR + HR group. ALR overexpression additionally had an impact on phosphorylation of Drp1 Ser637 during AKI. The inner membrane fusion protein, Optic Atrophy 1 (OPA1), was significantly increased whereas levels of outer membrane fusion proteins Mitofusin-1 and -2 (Mfn1, Mfn2) were not affected in the Lv-ALR + HR group, compared with the control group. Furthermore, the mTOR/4E-BP1 signaling pathway was highly activated in the Lv-ALR + HR group. ALR overexpression led to suppression of HR-induced apoptosis. Our collective findings indicate that ALR gene transfection alleviates mitochondrial injury, possibly through inhibiting fission and promoting fusion of the mitochondrial inner membrane, both of which contribute to reduction of HK-2 cell apoptosis. Additionally, fission processes are potentially mediated by promoting tubular cell survival through activating the mTOR/4E-BP1 signaling pathway.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.47-52
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• 1992

This study describes isolation and identification of a soil bacterium which is degradable of phosphinothricin and improvement of the isolated strain by using mutagenesis and spheroplast fusion. The experiment was performed to search for a possibility of development of a new strain which is both PPT-degradable and glyphosate-resistant by using interspecies cell fusion between the PPT-degrading bacterium. Pseudornonu.\ puucimohlis and a glyphosate -resistant strain, Pseudornonu.~ cc,pucicl. Auxotrophic mutants were obtained by the treatement of P. puucimohili.\ with ethylmethanosulfate, and used to cell fusion. Lysozyme and EDTA were used to spheroplast formation and regeneration rates :)f the spheroplast were 6.5'%1 in P. pauc.irnohili.\ and 8.8% in P.ci,j~u[,i(lr, espctively. Polyethylenglycol 5.000 was used to cell fusion as fusogen. The fusant\ulcorner F1. F2. F\ulcorner and F4 werc- obtained by the intra- and interspecies cell fusion. The fusant Fl of intraspecies cell fusion was higher to the wild type by 1 I'%l in PPT degrading ability, and the fusant F3 of inierspesis cell fusion developed plyphosatc-resistant and PPI-dcgrading ability which were propertics of two parental strains.

• Archives of Pharmacal Research
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• v.15 no.1
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• pp.30-34
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• 1992

In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were secreened. Among them, Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed between S. rimosus (N-cetylation function) and S. aureofaciens (p-hydroxylation function) and also between S. lividans and S. globisporus. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixed S rimosus ($pro^- \;his^-$) and S. aureofaciens ($ilv^-$) protoplasts with 40% (w/v) polythylene glycol 3350 (PEG) for 3 min gave $8.3\times10^{-7}$ of fusion frequency. Treatment of mixed S. lividans (pant-) and S. globisporus (leu-) protoplasts with 50% (w/v) PEG for 3 min at $30^\circ{C}$ gave $1.2\times10^{-6}$ of frequency. Among the fused strains, up to 40-50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts of S. auroefaciens (2% frequency) lost their p-hydroxylation function.

• YAKHAK HOEJI
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• v.35 no.1
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• pp.30-37
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• 1991

Streptomyces actuosus DMCJ-49 and Streptomyces minoensis DMCJ-144 produce the .alpha.-amylase inhibitor. Inerspecific protoplast fusion technique was used to increase the productivity of .alpha.-amylase inhibitor. Four auxotrophic mutants were obtained respectively from two strains by N-methyl-N'-nitor-N-nitrosoguanidine(3mg/ml) treatment. The optimum conditions for the protoplast formation of Streptomyces actuosus DMCJ-49 ade was as follows; 1.2% w/v of glycine, 3mg/ml of lysozyme, and 30 min of lysozyme treatment followed by 36 hr. incubation in the protop-last formation medium. In case of DMCJ-144-his those were 1.2%w/v, 3 mg/ml, 30 minutes and 60 hours, respectively. Regeneration was accomplished with hypertonic soft agar medium that contained 0.4M sucrose, 20mM CaCl$_2$, 50 mM MgCl$_2$ and low levels of phosphate. Fusion of protoplasts carrying different auxotrophic markers was achieved by treatment with polyethylene glycol. The optimum concentration of polyethylene glycol 1450 for the production of recombinants was 40%w/v. When the protoplasts was treated with 40% polyethylene glycol for 30 minutes, the frequency of recombinants was 6.5$\times$$10^{-3}$ and the $\alpha$-amylase inhibition activity of $ade^-his^-$ No. 4, which is the fusant with the most improved activity increased from 33 to 125 I.U./ml.

• Polymer(Korea)
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• v.32 no.6
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• pp.516-522
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• 2008

Biodegradable polymers have been used extensively as scaffolding materials to regenerate new tissues and the ingrowth of tissue have been reported to be dependent directly of the porosity, pore diameter, pore shape, and porous structure of the scaffold. In this study, porous poly (L-lactide-co-glycolide) (PLGA) scaffolds with five different pore sizes were fabricated to investigate the effect of pore sizes for AF tissue regeneration. Cellular viability and proliferation were assayed by MTT test. Hydroxyproline/DNA content of AF cells on each scaffold was measured. sGAG analyses were performed at each time point of 2 and 6 weeks. Scaffold seeded AF cells were implanted into the back of athymic nude mouse to observe the difference of formation of disc-like tissue depending on pore size in vivo. We confirmed that scaffold with $180{\sim}250{\mu}m$ pores displayed high cell viability in vitro and produced higher ECM than scaffold with other pore sizes in vivo.

• Applied Biological Chemistry
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• v.32 no.4
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• pp.435-440
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• 1989

To improve alkaline protease producing strain by protoplast fusion, a strain of Bacillus sp. was treated with $100{\mu}g/ml$ NTG (N-methyl-N'-itro-N-nitrosoguanidine) for 45 minutes and mutants of Bacillus subtilts $Arg^-,\;Try^-,\;His^-$ and $Ade^-$ were isolated. The frequency of protoplast formation was about 99%, when teas of exponential phase were treated with $200{\mu}g/ml$ lysozyme at $42^{\circ}C$ for $10{\sim}30$ minutes. In a regeneration medium containing 0.3M sodium succinate, 2.0% polyvinylpyrrolidone, 0.5% casamino acid, 10mM $MgCI_2$ and 20mM $CaCI_2$, regeneration frequency cf the isolated Bacillus subtilis strains was 25.2%. The fusion frequency between mutant was from $2.1{\times}10^{-5}$ to $8.1{\times}10^{-5}$ under optimum condition.

• Molecules and Cells
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• v.27 no.4
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• pp.403-408
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• 2009

Skeletal muscle regeneration is a highly orchestrated process initiated by activation of adult muscle satellite cells. Upon muscle injury, the inflammatory process is always accompanied by muscle regeneration. Leukotriene $B_4$ is one of the essential inflammatory mediators. We isolated and cultured primary satellite cells. RT-PCR showed that myoblasts expressed mRNA for $LTB_4$ receptors BLT1 and BLT2, and $LTB_4$ promoted myoblast proliferation and fusion. Quantitative real-time PCR and immunoblotting showed that $LTB_4$ treatment expedited the expression process of differentiation markers MyoD and M-cadherin. U-75302, a specific BLT1 inhibitor, but not LY2552833, a specific BLT2 inhibitor, blocked proliferation and differentiation of myoblasts induced by $LTB_4$, which implies the involvement of the BLT1 pathway. Overall, the data suggest that $LTB_4$ contributes to muscle regeneration by accelerating proliferation and differentiation of satellite cells.

• Polymer(Korea)
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• v.35 no.5
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• pp.378-384
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• 2011

Silk fibroin is a biocompatible and slowly biodegradable natural polymer. This natural polymer has excellent mechanical properties, non-toxicity, and non-immunogenic properties and has been demonstrated to support tissue regeneration. Also, gelatin is a natural material derived from collagen by hydrolysis and has an almost identical composition as that of collagen. Silk fibroin/gelatin scaffolds have been fabricated by using the freeze-drying method. To establish the scaffold manufacturing condition for silk fibroin and gelatin, we made scaffolds with various compositions of gelatin, glutaldehyde and silk fibroin. The silk fibroin/gelatin scaffolds were characterized using SEM, DSC, and water absorption ability tests. The cellular proliferation was evaluated by WST assay. These results suggested that a scaffold containing 8% of gelatin, 1% of glutaldehyde and 0.3 g of silk fibroin provided suitable characterstics for cell adhesion and proliferation. In conclusion, the silk fibroin/gelatin scaffold may serve as a potential cell delivery vehicle and a structural basis for tissue engineering.

• Applied Biological Chemistry
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• v.28 no.1
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• pp.13-18
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• 1985

To investigate the fusion of bacterial protoplasts, two auxotrophic mutants of Bacillus subtilis were isolated after treatment with nitrosoguanidine. Two auxotrophs were uracil-auxotroph and tryptophan-auxotroph. Back mutation frequencies of $ura^-$ and $trp^-$ -auxotrophs were $2.4{\times}10^{-8}$ and less than $1.0{\times}10^{-8}$, respectively. The optimal pH and temperature of protoplast formation with lysozyme were 6.5 and $30^{\circ}C$. The optimal lysozyme concentration was $200{\mu}g/ml$. The regeneration frequency of protoplast was 3.3%.