• Journal of Microbiology
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• v.40 no.1
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• pp.70-76
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• 2002

Proviral DNAs from HIV-1-infected CD4+ T cells (Molt/LAV cells) were amplified and detected in infected individual cells using polymerase chain reaction and in rifu hybridization. In this in situ PCR, three parameters were considered to achieve effective amplification and retention of amplificants inside the cells by making high molecular weight PCR products intracellularly, forming agarose matrix against the cells, and maintaining the appropriate PCR temperature profile. Over the cycles of ampliHcationl tailed primers with complementary overhanging sequences at their 5' sides manufactured high molecular weight products by using short primary products as a repeating unit. Agarose matrix could prevent the diffusion of the amplificants from the cells. Use of Thermanox coverslip inside the PCR tube offered target cells a similar temperature profile to that of conventional PCR in solution.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.167-171
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• 2004

Reduction in the leakage of the amplified PCR product out of cell is required for effective in situ PCR. For this purpose, primers with complementary tail sequences at their 5' sides were utilized to synthesize high molecular weight PCR products, but it is time-consuming and causes deterioration of cellular appearance with many PCR cycles. Therefore, it is required to optimize the PCR condition with minimal PCR cycles. To achieve the pur-pose, primer polymers were made without the target DNA in tube from nonspecific amplification with tailed primers and treated onto the fixed Molt/LAV cells on the glass slide for the 20 cycle-in situ PCR, in which the appropriate target signals were observed for the possible use of primer polymers in in situ PCR.

• Korean Journal of Microbiology
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• v.37 no.4
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• pp.294-298
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• 2001

Highly effective polymerase chain reaction (PCR) often brings about false positivity caused by contamination of the sample with target nucleic acids. To solve this problem, in situ PCR (ISPCR) has been developed and applied onto various tissue sections and suspension cultures. With combination of PCR and in situ hybridization, this method amplifies the nucleic acid targets in situ and detect the amplified products inside the cells over the background of various cell types. In order to amplify the nucleic acid targets inside the cells, permeabilisation of a sample is required for the entry of amplification reactants into a cell. Treatments of a sample for the purpose allow not only the entry of reactants into the cell but also the exit of amplification products out of the cell. As a means to reduce the leakage of the amplification products, two methods were applied to suspension cultures of HIV-infected Molt/LAV and U 1.1 cells, in which modified, tailed primers produced long linear amplificants whereas biotinylated dUTP instead of dTTP did bulky products.

• Korean Journal of Microbiology
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• v.39 no.3
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• pp.197-200
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• 2003

In order to establish effective in situ PCR on the glass slide using the conventional DNA thermal cycler, several parameters should be considered. These include full accessibility of PCR reagents into the cells, prevention of diffusing PCR products out of the cells, loss of PCR reagents by nonspecific adherence onto the glass slide, dryness of PCR reagents by heat, and heat conductivity from the heat block to the glass slide. Especially, to guarantee the full accessibility of PCR reagents to sample, relatively higher concentration of PCR reagents (particularly 4.5 mM of $Mg^{++}$) was required while 5 to 10 units/50 ${\mu}l$ reaction of Taq enzyme was enough as long as the step of pre-PCR incubation was included. Dryness of sample was prevented by addition of distilled water into the empty slots in the heat block, thereby providing the reproducible temperature-time profile of PCR. Observed temperature was lower than the programmed temperature by 3 to $4^{\circ}C$.

• Journal of Life Science
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• v.29 no.1
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• pp.90-96
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• 2019

The objective of this study was to evaluate the potential of Diospyros malabarica stem extract, a natural materials, in oral health material. With this aim in mind, thin layer chromatography (TLC), TLC-bioautography, high-performance liquid chromatography (HPLC), electrospray ionization-mass spectrometry (ESI-MS), scanning electron microscopy (SEM), and real-time qPCR were performed. The antibacterial activity of D. malabarica stem extract against Streptococcus mutans KCTC3065 was confirmed in an n-hexane fraction with low polarity. The molecular weight of the antibacterial compound was estimated to be 188 by ESI-MS analysis. The inhibitory effects of the extract on biofilm formation and gene expression related to biofilm formation of S. mutans were determined by SEM and real-time PCR analysis. The extract inhibited the formation of S. mutans biofilms at D. malabarica stem extract concentrations of 1 mg/ml, as shown by SEM. The real-time PCR analysis showed that the expression of the gtfC gene, which is associated with biofilm formation, was significantly decreased in a dose-dependent manner. Based on the above results, it can be concluded that D. malabarica stem extracts, a natural materials, can be used in oral health products to suppress the formation of biofilms by inhibiting tooth adhesion of S. mutans, a causative agent of dental caries.

• Journal of Life Science
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• v.24 no.6
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• pp.618-625
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• 2014

Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, making it less suitable than wheat for many food products such as bread and noodles. The high-molecular weight glutenin subunits (HMW-GS) of wheat play a crucial role in determining the processing properties of the wheat grain. This paper describes the development of marker-free transgenic rice plants expressing a wheat Glu-Dy10 gene encoding the HMG-GS from the Korean wheat cultivar 'Jokyeong' using Agrobacterium-mediated co-transformation. Two expression cassettes, consisting of separate DNA fragments containing Glu-1Dy10 and hygromycin phosphotransferase II (HPTII) resistance genes, were introduced separately into Agrobacterium tumefaciens EHA105 for co-infection. Each EHA105 strain harboring Glu-1Dy10 or HPTII was infected into rice calli at a 3: 1 ratio of Glu-1Bx7 and HPTII. Among 290 hygromycin-resistant $T_0$ plants, we obtained 29 transgenic lines with both the Glu-1Dy10 and HPTII genes inserted into the rice genome. We reconfirmed the integration of the Glu-1Dy10 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the Glu-1Dy10 in transgenic rice seeds were examined by semi-quantitative RT-PCR and Western blot analysis. The marker-free plants containing only the Glu-1Dy10 gene were successfully screened in the $T_1$ generation.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1386-1392
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• 2008

A gene (sll0158) putatively encoding a glycogen branching enzyme (GBE, E.C. 2.4.1.18) was cloned from Synechocystis sp. PCC6803, and the recombinant protein expressed and characterized. The PCR-amplified putative GBE gene was ligated into a pET-21a plasmid vector harboring a T7 promoter, and the recombinant DNA transformed into a host cell, E. coli BL21(DE3). The IPTG-induced enzymes were then extracted and purified using Ni-NTA affinity chromatography. The putative GBE gene was found to be composed of 2,310 nucleotides and encoded 770 amino acids, corresponding to approx. 90.7 kDa, as confirmed by SDS-PAGE and MALDI-TOF-MS analyses. The optimal conditions for GBE activity were investigated by measuring the absorbance change in iodine affinity, and shown to be pH 8.0 and $30^{\circ}C$ in a 50 mM glycine-NaOH buffer. The action pattern of the GBE on amylose, an $\alpha$-(1,4)-linked linear glucan, was analyzed using high-performance anion-exchange chromatography (HPAEC) after isoamylolysis. As a result, the GBE displayed $\alpha$-glucosyl transferring activity by cleaving the $\alpha$-(1,4)-linkages and transferring the cleaved maltoglycosyl moiety to form new $\alpha$-(1,6)-branch linkages. A time-course study of the GBE reaction was carried out with biosynthetic amylose (BSAM; $M_p{\cong}$8,000), and the changes in the branch-chain length distribution were evaluated. When increasing the reaction time up to 48 h, the weight- and number-average DP ($DP_w$ and $DP_n$) decreased from 19.6 to 8.7 and from 17.6 to 7.8, respectively. The molecular size ($M_p$, peak $M_w{\cong}2.45-2.75{\times}10^5$) of the GBE-reacted product from BSAM reached the size of amylose (AM) in botanical starch, yet the product was highly soluble and stable in water, unlike AM molecules. Thus, GBE-generated products can provide new food and non-food applications, owing to their unique physical properties.

• Journal of Nutrition and Health
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• v.52 no.6
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• pp.529-539
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• 2019

Purpose: Sprouts of evening primrose (Oenothera laciniata, OL) were reported to have high contents of flavonoids and potent antioxidant activity. This study examined the antioxidant and antiobesity activities of OL sprouts to determine if they could be a natural health-beneficial resource preventing obesity and oxidative stress. Methods: OL sprouts were extracted with 50% ethanol, evaporated, and lyophilized (OLE). The in vitro antioxidant activity of OLE was examined using four different tests. The antiobesity activity and in vivo antioxidant activity from OLE consumption were examined using high fat diet-induced obese (DIO) C57BL/6 mice. Results: The IC₅₀ for the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging and superoxide dismutase (SOD)-like activities of OLE were 26.2 ㎍/mL and 327.6 ㎍/mL, respectively. OLE exhibited the ferric reducing antioxidant power (FRAP) activity of 56.7 ㎍ ascorbic acid eq./mL at 100 ㎍/mL, and an increased glutathione level by 65.1% at 200 ㎍/mL compared to the control in the hUC-MSC stem cells. In an animal study, oral treatment with 50 mg or 100 mg of OLE/kg body weight for 14 weeks reduced the body weight gain, visceral fat content, fat cell size, blood leptin, and triglyceride levels, as well as the atherogenic index compared to the high fat diet control group (HFC) (p < 0.05). The blood malondialdehyde (MDA) level and the catalase and SOD-1 activities in adipose tissue were reduced significantly by the OLE treatment compared to HFC as well (p < 0.05). In epididymal adipose tissue, the OLE treatment reduced the mRNA expression of leptin, PPAR-γ and FAS significantly (p < 0.05) compared to HFC while it increased adiponectin expression (p < 0.05). Conclusion: OLE consumption has potent antioxidant and antiobesity activities via the suppression of oxidative stress and lipogenesis in DIO mice. Therefore, OLE could be a good candidate as a natural resource to develop functional food products that prevent obesity and oxidative stress.