• Journal of Veterinary Clinics
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• v.27 no.5
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• pp.508-513
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• 2010

Cytokines are important mediators of the immune response, and quantitating cytokine mRNA is a highly sensitive and attractive method for measuring cytokine production. The objective of the current study was to develop and validate a SYBR green quantitative real-time reverse transcriptase PCR (qRT-PCR) assay for measuring canine cytokine mRNA. The optimal annealing temperatures ($T_a$) of the designed primers were $62^{\circ}C$ for interleukin (IL)-$1{\beta}$, IL-6 and IL-10; $60^{\circ}C$ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tumor necrosis factor (TNF)-${\alpha}$; and $58^{\circ}C$ for high mobility group box 1 (HMGB1). Primer efficiencies of all primers calculated for standard curve samples were between 97.1% and 102.6%. No evidence of secondary structure or primer-dimer formation was seen via melt-curve analysis or gel electrophoresis. The developed qRT-PCR assays are highly specific and sensitive and can be used to quantify gene expression levels of canine cytokines.

• Journal of Life Science
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• v.25 no.6
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• pp.673-679
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• 2015

The culture conditions needed to maximize the production of laccase from Pholiota highlandensis mycelia were investigated. Among the tested media for laccase production, Coriolus versicolor medium (CVM; 2% dextrose, 0.4% peptone, 0.6% yeast extract, 0.046% KH₂PO₄, 0.1% K₂HPO₄, 0.05% MgSO₄·7H₂O) showed the highest activity for the enzyme. Then, to optimize culture conditions for laccase activity, the influences of various carbon, nitrogen, phosphorus, and inorganic salt sources in CVM were investigated. The optimum culture medium was 2% fructose, 0.4% peptone with 0.6% yeast extract, 0.05% NaH₂PO₄, and 0.05% MgSO₄·7H₂O as carbon, nitrogen, phosphorus, and inorganic salt sources, respectively. Several aromatic compounds in the medium enhanced laccase activity to varying degrees. Guaiacol induced maximum laccase production, yielding 114.1 U/ml laccase activity after cultivation for 11 days at 25℃. The optimum pH and temperature for laccase production were 8.0 and 35℃, respectively. Native polyacrylamide-gel electrophoresis (PAGE) followed by laccase-activity staining with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate was performed to identify the presence of laccase under the optimum conditions studied. Zymogram analysis of the supernatant culture showed an enzymatic band with a molecular mass of about 90 kDa.

• Journal of Life Science
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• v.25 no.2
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• pp.197-205
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• 2015

The comparative effects of the fibrinolytic action, antioxidative activity, and tyrosinase inhibition of Cordyceps militaris powder and fermented Cordyceps militaris powders were investigated using several microscopic organisms. The nutritional components such as phenolic compounds, flavonoids, and minerals were also measured. The total phenolic compounds and flavonoid concentrations were highest in the Cordyceps militaris powder fermented by Aspergillus oryzae. Major minerals were K, Ca, Mg, and Zn. Native polyacrylamide gel electrophoresis (native-PAGE) analysis of the total protein patterns of Cordyceps militaris powder and fermented Cordyceps militaris powders revealed slight varietal differences. Fibrinolytic activity was highest in the Cordyceps militaris powder fermented by Bacillus subtilis and Aspergillus kawachii. The DPPH radical scavenging activity was slightly stronger in the powder fermented by Monascus purpureus; however, these samples all exhibited a relatively low activity when compared with butylated hydroxytoluene (BHT). Tyrosinase inhibition activity was stronger in the powder fermented by Aspergillus oryzae than in unfermented powder. These results may provide basic data for understanding the biological activities and chemical characteristics of Cordyceps militaris powder fermented by several microscopic organisms for the development of functional foods.

• Journal of Life Science
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• v.22 no.11
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• pp.1523-1528
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• 2012

In a previous proteomic study, heat shock protein beta 1 (HSPB1) was detected as differentially expressed protein in longissimus thoracis between low (grade 3) and high (grade 1++) meat quality groups by 2DE gel electrophoresis. The present study investigated an association of HSPB1 expression at the level of gene and protein with Warner-Bratzler shear force (WBS) measured in 20 Hanwoo steers. An analysis of variance (ANOVA) between expression values and WBS showed that WBS was affected by HSPB1 expression (p<0.05). The expression (at both gene and protein level) of the HSPB1 was 2 times higher in the low WBS group than that in the high WBS group (p<0.01). This result suggests that the HSPB1 gene may be a candidate gene associated with tenderness in longissimus thoracis of Korean cattle.

• Journal of Life Science
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• v.18 no.3
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• pp.336-343
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• 2008

Histone deacetylases (HDACs) inhibitors have emerged as the accessory therapeutic agents for various human cancers, since they can block the activity of specific HDACs, restore the expression of some tumor suppressor genes and induce cell differentiation, cell cycle arrest and apoptosis in vitro and in vivo. In the present study, we investigated that the effect of trichostatin A (TSA), an HDAC inhibitor, on the cell growth and apoptosis, and its effect on the nuclear factor-kappaB $(NF-{\kappa}B)$ activity in 267B1 human prostate epithelial cells. Exposure of 267B1 cells to TSA resulted in growth inhibition and apoptosis induction in and dose-dependent manners as measured by fluorescence microscopy, agarose gel electrophoresis and flow cytometry analysis. TSA treatment inhibited the levels of IAP family members such as c-IAP-1 and c-IAP-2 and induced the proteolytic activation of caspase-3, -8 and -9, which were associated with concomitant degradation of poly (ADP-ribose)-polymerase, ${\beta}-catenin$ and laminin B proteins. The increase in apoptosis by TSA was connected with the translocation of $NF-{\kappa}B$ from cytosol to nucleus, increase of the DNA binding as well as promoter activity of $NF-{\kappa}B$, and degradation of cytosolic inhibitor of KappaB $(I{\kappa}B)-{\alpha}$ protein. We therefore concluded that TSA demonstrated anti-proliferative and apoptosis-inducing effects on 267B1 cells in vitro, and that the activation of caspases and $NF-{\kappa}B$ may play important roles in its mechanism of action. Although further studies are needed, these findings provided important insights into the possible molecular mechanisms of the anti-cancer activity of TSA.

• Journal of Life Science
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• v.26 no.9
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• pp.1007-1014
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• 2016

A highly efficient, rapid, and reliable multiplex polymerase chain reaction based method for distinguishing ten species of genus Cynoglossus (C. senegalensis, C. abbreviates, C. macrolepidotus, C. arel, C. semilaevis, C. interruptus, C. joyneri, C. lingua, C. robustus, and C. monodi) is described. The species-specific primer sets were designed base on the cytochrome oxidase subunit I gene (1,500 bp). The optimal PCR conditions and primers were selected for ten of Cynoglossus species to determine target base sequences using single PCR. Multiplex PCR using the ten pairs of primers either specifically amplified a DNA fragment of a unique size or failed, depending on each species DNA. The length of amplification fragment of 208 bp for C. senegalensis, 322 bp for C. abbreviates, 493 bp for C. macrolepidotus, 754 bp for C. arel, 874 bp for C. semilaevis, 952 bp for C. interruptus, 1,084 bp for C. joyneri, 1,198 bp for C. lingua, 1,307 bp for C. robustus, and 1,483 bp for C. monodi with the species-specific primers, visualized by agarose gel electrophoresis, allowed perfectly distinction of the Cynoglossus species. The multiplex PCR assay can be easily performed on multiple samples and attain final results in less than 6 hours. This technique should be a useful addition to the molecular typing tools for the tentative identification of Cynoglossus species.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.120-124
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• 2016

A certain size of DNA (28-29 kb long) to be packaged into P2-size head and the mutation in sid gene of bacteriophage P4 are the major factors to overcome "P2 sir-associated helper inefficiency". To clarify whether the presence of sid mutation is essential to overcome "P2 sir-associated helper inefficiency" or not, we tested the P4 derivative, P4 delRI::kmr, which is $sid^+$ and whose genome size supposed to be 28.5 kb long in the case of being packaged into $P2_{sir3}$-sized large head. As P4 delRI::kmr showed the low EOP with P2 sir3 lysogen, P4 delRI::kmr phage stock was prepared in P2 sir3 lysogen host to increase the EOP with P2 sir3 lysogen. Through this process, P4 delRI::kmr had been adapted for P2 sir3 lysogen. With a CsCl buoyant equilibrium density gradient experiment and gel electrophoresis of the isolated DNA, it was evident that the adaptation of P4 delRI::kmr for P2 sir3 lysogen was caused by the conformational change of DNA to be packaged into large head. The burst size determination experiments with P4 delRI::kmr phage stock adapted for P2 sir3 lysogen and normal P4 delRI::kmr phage stock showed that not the sid mutation but the size of DNA to be packaged (28-29 kb long) was essential to overcome "P2 sir-associated helper inefficiency".

• Food Science of Animal Resources
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• v.36 no.6
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• pp.779-786
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• 2016

This study aimed to evaluate the prevalence of Listeria monocytogenes on livestock farms in Korea and determine their serotypes and genetic correlations. Twenty-five livestock farms in Korea (central: 15, south west: 7, south east: 3) were visited 2-3 times, and 2,018 samples (feces: 677, soil: 680, silage: 647, sludge: 14) were collected. Samples were enriched in LEB (Listeria enrichment broth) and Fraser broth media, and then plated on Palcam agar. The isolates were identified by PCR and 16S rRNA gene sequencing. Then, the sero-types, presence of virulence genes (actA, inlA, inlB, plcB, and hlyA), and antibiotic resistance were determined. Genetic correlations among the isolates were evaluated by analyzing the restriction digest pattern with AscI. Of the 2,018 samples, only 3 (0.15%) soil samples (FI-1-FI-3) from 1 farm in the south east region were positive for L. monocytogenes. Based on biochemical tests and multiplex PCR, the serotype of the isolates were 4ab (FI-1 and FI-3) and 3a (FI-2), which are not common in foodborne L. monocytogenes. The 3a sero-type isolate was positive for all tested virulence genes, whereas the 4ab serotype isolates were only positive for hlyA, actA, and inlA. The isolates were resistant to all 12 tested antibiotics, especially FI-3. The genetic correlations among the isolates were 100% for those of the same serotype and 26.3% for those of different serotypes. These results indicate that the prevalence of L. monocytogenes on livestock farms in Korea is low; however, the isolates are pathogenic and antibiotic resistant.

• Food Science of Animal Resources
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• v.40 no.5
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• pp.689-698
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• 2020

The aim of study was to scrutinize the physicochemical and protein profile of milk obtained from local Pakistani breeds of milch animals such as Nilli-Ravi buffalo, Sahiwal cow, Kajli sheep, Beetal goat and Brela camel. Physicochemical analysis unveiled maximum number of total solids and protein found in sheep and minimum in camel. Buffalo milk contains the highest level of fat (7.45%) while camel milk contains minimum (1.94%). Ash was found maximum in buffalo (0.81%) and sheep (0.80%) while minimum in cow's milk (0.71%). Casein and whey proteins were separated by subjecting milk to isoelectric pH and then analyzed through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed heterogeneity among these species. Different fractions including α_S1, α_S2, κ-casein, β-casein and β-lactoglobulen (β-Lg) were identified and quantitatively compared in all milk samples. Additionally, this electrophoretic method after examining the number and strength of different protein bands (α_S1, α_S2, β-CN, α-LAC, BSA, and β-Lg, etc.), was helpful to understand the properties of milk for different processing purposes and could be successfully applied in dairy industry. Results revealed that camel milk was best suitable for producing allergen free milk protein products. Furthermore, based on the variability of milk proteins, it is suggested to clarify the phylogenetic relationships between different cattle breeds and to gather the necessary data to preserve the genetic fund and biodiversity of the local breeds. Thus, the study of milk protein from different breed and species has a wide range of scope in producing diverse protein based dairy products like cheese.

• The Journal of Korean Medicine
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• v.26 no.1 s.61
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• pp.174-186
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• 2005

Objectives : The water extract of Samul-tang (SMT) has traditionally been used for treatment of ischemic heart and brain damage in oriental medicine. However, little is known about the mechanism by which the water extract of SMT rescues cells from these damages. Methods: This study was designed to investigate the protective mechanisms of SMT on oxidative stress-induced toxicity in H9c2 cardiomyoblast cells. Treatment with $H_2O_2$ markedly induced death of H9c2 cardiomyoblast cells in a dose-dependent manner. Results: The characteristics of H20z-induced death of H9c2 showed apparent apoptotic features such as DNA fragmentation and morphological change. However, SMT significantly reduced both H202-induced cell death and morphological change. The decrease of Bc-2 expression by High were inhibited by SMT. In addition, the increase of Bax expression was also inhibited by SMT. The cotreatment of SMT and $H_2O_2$ in H9c2 cells also induced the phosphorylation of ERK in a time-dependent manner. Moreover, PD98059, a specific inhibitor of ERK1/2 attenuated the protective effects of SMT on $H_2O_2-induced$ toxicity in H9c2 cardiomyoblast cells. These results suggest that both ERK1/2 signaling pathways play important roles in the protective effects of SMT on $H_2O_2-induced$ apoptotic death of H9c2 cells. Also, the expression profile of proteins in $H_2O_2$ cardiomyoblast cells were screened by using two-dimensional (2-D) gel electrophoresis. Among 300 spots resolved in 2-D gels, the comparison of control versus apoptosis cells revealed that signal intensity of 17 spots increased and 11 spots decreased. Conclusions: Taken together, this study suggests that the protectiw effects of the water extract of SMT against oxidative damages may be mediated by the modulation of Bc1-2 and Bax expression via the regulation of the ERK signaling pathway.