• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.187-195
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• 2020

To understand the molecular mechanism involved in the survivability of cold-tolerant lactic acid bacteria was of great significance in food processing, since these bacteria play a key role in a variety of low-temperature fermented foods. In this study, the cold-stress response of probiotic Lactobacillus plantarum K25 isolated from Tibetan kefir grains was analyzed by iTRAQ proteomic method. By comparing differentially expressed (DE) protein profiles of the strain incubated at 10℃ and 37℃, 506 DE proteins were identified. The DE proteins involved in carbohydrate, amino acid and fatty acid biosynthesis and metabolism were significantly down-regulated, leading to a specific energy conservation survival mode. The DE proteins related to DNA repair, transcription and translation were up-regulated, implicating change of gene expression and more protein biosynthesis needed in response to cold stress. In addition, two-component system, quorum sensing and ABC (ATP-binding cassette) transporters also participated in cell cold-adaptation process. These findings provide novel insight into the cold-resistance mechanism in L. plantarum with potential application in low temperature fermented or preserved foods.

• Journal of Pharmaceutical Investigation
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• v.40 no.3
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• pp.139-153
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• 2010

Nitrosative stress is defined as pathophysiological conditions that are related to covalent modifications of proteins by nitration/nitrosylation by forms of nitrogen oxide ($NO_x$), leading to DNA damage, ultimately, cell death. This type of stress condition appears to be associated with a number of disease states, including diabetes, inflammation and neurodegenerative diseases. Since these pathological conditions are frequently chronic in nature and, thus, require long-term treatment, changes in pharmacokinetics are likely to affect the therapy. Transporters are membrane proteins that facilitate the movement of substrates, including drugs, across plasma membranes of epithelial / endothelial cells. Since it is now increasingly evident that transporters are pharmacokinetically significant, functional alteration of transporters by this stress condition may have therapeutic relevance. In this review, experimental techniques that are used to study both in vivo and in vitro nitrosative stress are summarized and discussed, along with available literature information on the functional implication of transporters under conditions of nitrosative stress conditions. In the literature, both functional induction and impa irment were apparently present for both drug transporter families [i.e., ATP-binding cassette (ABC) and solute carrier families (SLC)]. Furthermore, a change in the function of a certain transporter appears to have temporal dependency by impairment in the early phase of nitrosative stress and induction thereafter, suggesting that the role of nitrosative stress is complex in terms of functional implications of the transporters. Although the underlying mechanisms for these alterations are not fully understood, protein nitration/nitrosylation appears to be involved in the functional impairment whereas transcript factor(s) activated by nitrosative stress may play a role, at least in part, in functional induction. Interestingly, functional induction under conditions of nitrosative stress has not been observed for SLC transporters while such impairment has been documented for both ABC and SLC transporters. Further investigations appear to be necessary to fully delineate the underlying reasons for these differences on the impact and importance of nitrosative stress conditions.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1330-1335
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• 2011

N-Acyl homoserine lactones (AHLs) serve as the vital quorum-sensing signals that regulate the virulence of the pathogenic bacterium Erwinia carotovora. In the present study, an approach to efficiently restrain the pathogenicity of E. carotovora-induced soft rot disease is described. Bacillus thuringiensis-derived N-acyl homoserine lactonase (AiiA) was projected onto the surface of Pseudomonas putida cells, and inoculation with both strains was challenged. The previously identified N-terminal moiety of the ice nucleation protein, InaQ-N, was applied as the anchoring motif. A surface display cassette with inaQ-N/aiiA was constructed and expressed under the control of a constitutive promoter in P. putida AB92019. Surface localization of the fusion protein was confirmed by Western blot analysis, flow cytometry, and immunofluorescence microscopy. The antagonistic activity of P. putida MB116 expressing InaQ-N/AiiA toward E. carotovora ATCC25270 was evaluated by challenge inoculation in potato slices at different ratios. The results revealed a remarkable suppressing effect on E. carotovora infection. The active component was further analyzed using different cell fractions, and the cell surface-projected fusion protein was found to correspond to the suppressing effect.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.784-788
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• 2007

Growth hormone-releasing hormone (GHRH), a hypothalamic neuropeptide can stimulate the growth hormone secretion from the anterior pituitary. In this study, a porcine GHRH expression plasmid pHC-GHRH was used to enhance growth performance through ectopic expressions in muscle tissues of rats. Rats injected with the plasmid of pHC-GHRH and pCMV-GHRH exhibited cumulative weight gains 6.4% and 1% greater than controls. During a 5-day period, significant weight gain differences were observed as follows compared with that of control: during 5-10 days post-injection (DPI) period, the group pHC-GHRH on average 14.5% heavier than controls, $40.73{\pm}0.88$ g vs. $35.57{\pm}1.23$ g (p = 0.0023); during 10-15 DPI period, the group pHC-GHRH on average 13.6% heavier than controls, $37.49{\pm}2.85$ g vs. $33.00{\pm}1.56$ g (p = 0.0146); during 15-20 DPI period, the group pHC-GHRH on average 17.8% heavier than controls, $25.64{\pm}1.39$ g vs. $21.77{\pm}1.27$ g (p<0.05). In addition, plasmids-treated rats maintained higher serum IGF-I than controls. Significant differences of IGF-I were observed on 13 DPI and on 40 DPI in pHC-GHRH group compared with that of controls. This was accomplished through the use of an improved expression cassette that included the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.5-kilobase portion of porcine ${\alpha}$-skeletal muscle actin promoter.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.248-254
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• 2008

Candida magnoliae, an osmotolerant and erythritol producing yeast, prefers D-fructose to D-glucose as carbon sources. For the investigation of the fructophilic characteristics with respect to sugar transportation, a sequential extraction method using various detergents and ultracentrifugation was developed to isolate cellular membrane proteins in C. magnoliae. Immunoblot analysis with the Pma1 antibody and two-dimensional electrophoresis analysis coupled with MS showed that the fraction II was enriched with membrane proteins. Eighteen proteins out of 36 spots were identified as membrane or membrane-associated proteins involved in sugar uptake, stress response, carbon metabolism, and so on. Among them, three proteins were significantly upregulated under the fructose supplying conditions. The hexose transporter was highly homologous to Ght6p in Schizosaccharomyces pombe, which was known as a predominant transporter for the fructose uptake of S. pombe because it exhibited higher affinity to D-fructose than D-glucose. The physicochemical properties of the ATP-binding cassette transporter and inorganic transporter explained their direct or indirect associations with the fructophilic behavior of C. magnoliae. The identification and characterization of membrane proteins involved in sugar uptake might contribute to the elucidation of the selective utilization of fructose to glucose by C. magnoliae at a molecular level.

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1098-1105
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• 2017

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1719-1727
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• 2014

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

• Mycobiology
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• v.43 no.1
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• pp.31-36
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• 2015

We have previously isolated ${\varepsilon}$-COP, the ${\alpha}$-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of ${\varepsilon}$-COP, the $aneA^+$ gene for ${\varepsilon}$-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the ${\varepsilon}$-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of ${\varepsilon}$-COP did not rescue the thermo-sensitive growth defect of the ${\alpha}$-COP mutant at $42^{\circ}C$. Together, these data show that ${\varepsilon}$-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.

• Imaging Science in Dentistry
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• v.34 no.2
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• pp.63-67
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• 2004

Purpose: To make a focal trough (image layer) for an average maxillary dental arch of 6-year-old korean in panoramic radiography. Materials and Methods : Phantom for the maxillary dental arch was designed using intercanine width, intermolar width, tooth size, and interdental spacing to record the data of 6-year-old child. The characteristics of pre-corrected panoramic machine (for adult) was evaluated using the phantom, resolution test pattern for margin of the image layer, and metal ball for the center of the image layer. Panoramic image layer of the child was developed by means of decreasing the speed of film-cassette and positioning the phantom backwards, and then the characteristics of post-corrected panoramic machine (for child) were reevaluated. Results: At post-corrected panoramic image layer, beam projection angles at all interdental areas increased for about 2.6-3.8°, the position of the image layer was shifted toward the rotation center for about 2.5 mm at the deciduous central incisior area. The width of image layer decreased at all areas. Conclusion : Increased beam projection angle will reduce the disadvantage of tooth overlap, and the same form between the center of the image layer and dental arch will improve image resolution.

• Journal of Life Science
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• v.20 no.2
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• pp.176-182
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• 2010

Magnaporthe oryzae is destructive plant-pathogenic fungus and causes rice blast. The pathogen uses several mechanisms to circumvent the inhibitory actions of fungicides. ATP-binding cassette (ABC) transporters are known to provide protection against toxic compounds in the environment. PC facilitated bioinformatic analysis, particularly with respect to accessing and extracting database information and domain identification. We predicted ABC transporter genes from the M. oryzae genome sequence with computation and bioinformatics tools. A total of thirty three genes were predicted to encode ABC transporters. Three of thirty three putative genes corresponded to three known ABC transporter genes (ABC1, ABC2 and ABC3). Copy numbers of the ABC transporter genes were proven by Southern blot analysis, which revealed that twenty genes tested exist as a single copy. We amplified the DNA complementary to RNA corresponding to eleven of these by reverse transcriptase polymerase chain reaction.