• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.183-190
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• 1994

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.739-747
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• 2018

The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJ-GrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJ-GrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10,c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.5055-5061
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• 2014

In this study, an anti-oxidant and anti-tumor protein Latcripin-3 of Lentinula edodes C91-3 was expressed in Escherichia coli. for the first time. According to the cDNA library, the full-length gene of Latcripin-3 was cloned by the methods of 3'-full rapid amplification of cDNA Ends (RACE) and 5'-full RACE. The structural domain gene of Latcripin-3 was inserted into the pET32 a(+). The functional protein of Latcripin-3 was expressed in Rosetta-gami (DE3) E. coli, evaluated by Western blotting and mass spectrometry. DPPH testing showed that the protein Latcripin-3 can scavenge free radicals remarkably well. The activity of functional protein Latcripin-3 on A549 cells was studied with flow cytometry and the MTT method. The MTT assay results showed that there was a decreases in cell viability in a dose-dependent and time-dependent manner in protein Latcripin-3 treated groups. Flow cytometry demonstrated that Latcripin-3 can induce apoptosis and block S phase dramatically in human A549 lung cancer cells as compared to the control group. At the same time, the cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. This research offers new insights and advantages for identifying anti-oxidant and anti-tumor proteins.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.6
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• pp.648-654
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• 1998

Twenty-four Javanese thin-tail ewes (11, 9, and 4 ewes giving birth to 1, 2, and 3 lambs, respectively) with similar body weight and age at breeding were used to study serum progesterone concentrations during pregnancy, milk production during lactation, and mammary gland indices at the end of lactation (3 months postpartum). The results of the experiment showed that averages serum progesterone concentrations during pregnancy in the ewes giving birth to twin and triplet lambs were higher (p < 0.01) than those giving birth to a single lamb. Ewes giving birth to 3 lambs had higher (p < 0.01) mammary dry fat-free tissue (DFFT) (by 31 and 34%), DNA concentration (by 25 and 16%) and RNA concentration (by 29 and 16%) at the end of lactation than those giving birth to 1 and 2 lambs. There was no difference in mammary collagen, protein and glycogen concentrations at the end of lactation among litter sizes. Ewes giving birth to 3 lambs had higher (p < 0.01) total mammary DNA content (by 64 and 61%) and RNA content (by 69 and 53%) at the end of lactation than those giving birth to 1 and 2 lambs. There was no difference in total mammary collagen, protein and glycogen contents at the end of lactation among litter sizes. Even though ewes with higher litter size had numerically higher milk production, there was no significant difference in milk production per 4 h among litter sizes. The results of the experiment indicated that ewes having higher litter size had greater mammary cell number and synthetic activities at the end of lactation. The results suggested that ewes with higher progesterone concentrations and better developed mammary glands during pregnancy could maintain higher cell number and activities throughout lactation.

• Progress in Medical Physics
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• v.11 no.2
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• pp.157-165
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• 2000

Purpose: For estimation of yields of l)NA damages induced by radiation and enhanced by oxygen, a mathematical model was used and tested. Materials and Methods: Reactions of the products of water radiolysis were modeled as an ordinary time dependant equations. These reactions include formation of radicals, DNA damage, damage repair, restitution, and damage fixation by oxygen and H-radical. Several rate constants were obtained from literature while others were calculated by fitting an experimental data. Sensitivity studies were performed changing the chemical rate constant at a constant oxygen number density and varying the oxygen concentration. The effects of oxygen concentration as well as the damage fixation mechanism by oxygen were investigated. Oxygen enhancement ratio(OER) was calculated to compare the simulated data with experimental data. Results: Sensitivity studies with oxygen showed that DNA survival was a function of both oxygen concentration and the magnitude of chemical rate constants. There were no change in survival fraction as a function of dose while the oxygen concentration change from 0 to 1.0 x 10$^{7}$ . When the oxygen concentration change from 1.0 $\times$ 107 to 1.0 $\times$ 101o, there was significant decrease in cell survival. The OER values obtained from the simulation study were 2.32 at 10% cell survival level and 1.9 at 45% cell survival level. Conclusion: Sensitivity studies with oxygen demonstrated that the experimental data were reproduced with the effects being enhanced for the cases where the oxygen rate constants are largest and the oxygen concentration is increased. OER values obtained from the simulation study showed good agreement for a low level of cell survival. This indicated that the use of the semi-empirical model could predict the effect of oxygen in cell killing.

• Proceedings of the Korean Vacuum Society Conference
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• 2012.02a
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• pp.549-549
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• 2012

Early detection of cancer biomarkers in the blood is of vital importance for reducing the mortality and morbidity in a number of cancers. From this point of view, immunosensors based on nanowire (NW) and carbon nanotube (CNT) field-effect transistors (FETs) that allow the ultra-sensitive, highly specific, and label-free electrical detection of biomarkers received much attention. Nevertheless 1D nano-FET biosensors showed high performance, several challenges remain to be resolved for the uncomplicated, reproducible, low-cost and high-throughput nanofabrication. Recently, two-dimensional (2D) graphene and reduced GO (RGO) nanosheets or films find widespread applications such as clean energy storage and conversion devices, optical detector, field-effect transistors, electromechanical resonators, and chemical & biological sensors. In particular, the graphene- and RGO-FETs devices are very promising for sensing applications because of advantages including large detection area, low noise level in solution, ease of fabrication, and the high sensitivity to ions and biomolecules comparable to 1D nano-FETs. Even though a limited number of biosensor applications including chemical vapor deposition (CVD) grown graphene film for DNA detection, single-layer graphene for protein detection and single-layer graphene or solution-processed RGO film for cell monitoring have been reported, development of facile fabrication methods and full understanding of sensing mechanism are still lacking. Furthermore, there have been no reports on demonstration of ultrasensitive electrical detection of a cancer biomarker using the graphene- or RGO-FET. Here we describe scalable and facile fabrication of reduced graphene oxide FET (RGO-FET) with the capability of label-free, ultrasensitive electrical detection of a cancer biomarker, prostate specific antigen/${\alpha}$ 1-antichymotrypsin (PSA-ACT) complex, in which the ultrathin RGO channel was formed by a uniform self-assembly of two-dimensional RGO nanosheets, and also we will discuss about the immunosensing mechanism.

• Microbiology and Biotechnology Letters
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• v.23 no.1
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• pp.104-109
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• 1995

The gene responsible for dechlorination of 4-chlorobenzoate (4CBA) was cloned in E. coli XL1-Blue from Pseudomonas sp. DJ-12. The cloned cell of E. coli Cjl had the hybrid pBluescript SK(+) plasmid, into which about 9.5 kb genomic DNA fragment of PseudOmonas sp. DJ-12 was inserted. The subclone of pCJlOl was constructed by inserting the 3.4 kb EcoRI-HindIII fragment of pCJl into the vector. Those cloned cells could be simply selected by halo formation around the colonies which was the precipitate of AgCl produced by reaction of AgNO$_{3}$ and chloride ion liberated by bacterial dechlorination of 4CBA- Such a plate assay method was standardized by the procedure that the colonies grown for 2 days on the Cl$^{-}$-free plate medium containing 1 mM 4CBA were flooded with 0.1 M AgNO$_{3}$ solution.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.9
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• pp.4199-4204
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• 2016

Cancer is a complex disease caused by a progressive accumulation of multiple genetic mutations. Consumption of fruits is associated with lower risk of several cancers, which is mainly associated to their phytochemical content. The use of functional foods and chemopreventive compounds seems to contribute in this process, acting by mechanisms of antioxidant, anti-inflammatory, anti-angiogenic and hormonal. The Psidium Guajava has high potential functional related to pigments who are involved in the process of cancer prevention by having antioxidant activity. The aim of the present review is to expose some chemical compounds from P. Guajava fractions and their association with anti-carcinogenic function. The evidences supports the theory of anticancer properties of P. Guajava, although the mechanisms are still not fully elucidated, but may include scavenging free radicals, regulation of gene expression, modulation of cellular signalling pathways including those involved in DNA damage repair, cell proliferation and apoptosis.

• Journal of Applied Biological Chemistry
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• v.50 no.4
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• pp.221-226
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• 2007

The high molecular-weight mucilage extracted and purified from cactus fruit of Opuntia ficus-indica var. Saboten was degraded by the cell-free culture filtrate of a fungus isolated from soil. TLC analysis of the polymeric mucilage after incubation with the fungal culture filtrate confirmed its degradation. When the degradation products were tested for their qualitative reactions with ninhydrin and phenol-sulfuric acid, only phenol-sulfuric acid gave positive development, and ninhydrin did not show any observable color reaction. This coloring reaction suggested the presence of a carbohydrate without an amino group within the mucilage. Analyses by HPLC and liquid gel permeation chromatography on sephadex G-100 also provided additional information on degradation of the mucilage by the fungal culture filtrate. The sequences of ITS-5.8S rDNA from the fungal isolate that was cultivated for the preparation of mucilage-degrading enzyme showed 99% similarity to those of Pestalotiopsis aquatica.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1468-1471
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• 2006

The last step in L-carnitine biosynthesis in eukaryotic organisms is mediated by ${\gamma}$-butyrobetaine hydroxylase (EC1.14.11.1), a dioxygenase that converts ${\gamma}$-butyrobetaine to L-carnitine. This enzyme was previously identified from rat liver and humans, and the peptide sequence of human ${\gamma}$-butyrobetaine hydroxylase was used to search the Neurospora crassa genome database, which led to an identification of an open reading frame (ORF) consisting of 1,407 bp encoding a polypeptide of 468 amino acids. When this protein was expressed in Saccharomyces cerevisiae, the crude cell-free extract exhibited ${\gamma}$-butyrobetaine hydroxylase activity.