• Mass Spectrometry Letters
• /
• 제9권1호
• /
• pp.24-29
• /
• 2018

DC23, a triazolothione resorcinol analogue, is known to inhibit heat shock protein 90 and pyruvate dehydrogenase kinase which are up-regulated in cancer and diabetes, respectively. This study was performed to elucidate the metabolism of DC23 in human liver microsomes (HLMs). HLMs incubated with DC23 in the presence of uridine 5'-diphosphoglucuronic acid (UDPGA) and/or ${\beta}$-nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the formation of four metabolites, M1-M4. M1 was identified as DC23-N-Oxide, on the basis of LC-MS/MS analysis. DC23 was further metabolized to its glucuronide conjugates (M2, M3, and M4). In vitro metabolic stability studies conducted with DC23 in HLMs revealed significant glucuronide conjugation with a $t_{1/2}$ value of 1.3 min. The inhibitory potency of DC23 on five human cytochrome P450s was also investigated in HLMs. In these experiments, DC23 inhibited CYP2C9-mediated tolbutamide hydroxylase activity with an $IC_{50}$ value of $8.7{\mu}M$, which could have implications for drug interactions.

• Asian-Australasian Journal of Animal Sciences
• /
• 제32권5호
• /
• pp.648-656
• /
• 2019

Objective: An experiment was conducted to determine the effect of reduced energy density of close-up diets on metabolites, lipolysis and gluconeogenesis in cows during the transition period. Methods: Thirty-nine Holstein dry cows were blocked and assigned randomly to three groups, fed a high energy density diet (HD, 1.62 Mcal of net energy for lactation $[NE_L]/kg$ dry matter [DM]), a medium energy density diet (MD, $1.47Mcal\;NE_L/kg\;DM$), or a low energy density diet (LD, $1.30Mcal\;NE_L/kg\;DM$) prepartum; they were fed the same lactation diet to 28 days in milk (DIM). All the cows were housed in a free-stall barn and fed ad libitum. Results: The reduced energy density diets decreased the blood insulin concentration and increased nonesterified fatty acids (NEFA) concentration in the prepartum period (p<0.05). They also increased the concentrations of glucose, insulin and glucagon, and decreased the concentrations of NEFA and ${\beta}-hydroxybutyrate$ during the first 2 weeks of lactation (p<0.05). The plasma urea nitrogen concentration of both prepartum and postpartum was not affected by dietary energy density (p>0.05). The dietary energy density had no effect on mRNA abundance of insulin receptors, leptin and peroxisome proliferator-activated $receptor-{\gamma}$ in adipose tissue, and phosphoenolpyruvate carboxykinase, carnitine palmitoyltransferase-1 and peroxisome proliferator-activated $receptor-{\alpha}$ in liver during the transition period (p>0.05). The HD cows had higher mRNA abundance of hormone-sensitive lipase at 3 DIM compared with the MD cows and LD cows (p = 0.001). The mRNA abundance of hepatic pyruvate carboxy-kinase at 3 DIM tended to be increased by the reduced energy density of the close-up diets (p = 0.08). Conclusion: The reduced energy density diet prepartum was effective in controlling adipose tissue mobilization and improving the capacity of hepatic gluconeogenesis postpartum.

• Animal Bioscience
• /
• 제35권1호
• /
• pp.96-104
• /
• 2022

Objective: Sarcoplasmic proteins include proteins that play critical roles in biological processes of living organisms. How seasons influence biological processes and meat quality of postmortem muscles through the regulation of protein phosphorylation remain to be investigated. In this study, the phosphorylation of sarcoplasmic proteins in pork longissimus muscle was investigated in four seasons. Methods: Sarcoplasmic proteins were extracted from 40 pork carcasses (10 for each season) and analyzed through ProQ Diamond staining for phosphorylation labeling and Sypro Ruby staining for total protein labeling. The pH of muscle, contents of glycogen and ATP were measured at 45 min, 3 h, and 9 h postmortem and the water (P_2b, P₂₁, and P₂₂) was measured at 3 h and 9 h. Results: A total of 21 bands were detected. Band 8 (heat shock cognate 71 kDa protein; heat shock 70 kDa protein 1B) had higher phosphorylation level in summer than that in other seasons at 45 min postmortem. The phosphorylation levels of 3 Bands were significantly different between fast and normal pH decline groups (p<0.05). The phosphorylation levels of 4 bands showed negative associations with immobilized water (P₂₁) and positive association with free water (P₂₂). Conclusion: The phosphorylation levels of sarcoplasmic proteins involved in energy metabolism and heat stress response at early postmortem time differed depending on the seasons. These proteins include heat shock protein 70, pyruvate kinase, phosphoglucomutase-1, glucose-6-phosphate isomerase, and carbonic anhydrase 3. High temperatures in summer might result in the phosphorylation of those proteins, leading to pH decline and low water holding capacity.

• Animal Bioscience
• /
• 제35권8호
• /
• pp.1162-1173
• /
• 2022

Objective: This study aimed to investigate the fermentation profiles, bacterial community and predicted metabolic characteristics of Sudangrass (Sorghum sudanense Stapf.) during ensiling. Methods: First-cutting Sudangrass was harvested at the vegetative stage and ensiled in laboratory-scale silos (1 L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30, and 60 days of ensiling, respectively. The bacterial communities on day 3 and 60 were assessed through high-throughput sequencing technology, and 16S rRNA-gene predicted functional profiles were analyzed according to the Kyoto encyclopedia of genes and genomes using Tax4Fun. Results: The Sudangrass silages showed good fermentation quality, indicated by higher lactic acid contents, and lower pH, butyric acid and ammonia nitrogen contents. The dominant genus Lactococcus on day 3 was replaced by Lactobacillus on day 60. The metabolism of amino acid, energy, cofactors and vitamins was restricted, and metabolism of nucleotide and carbohydrate was promoted after ensiling. The 1-phosphofructokinase and pyruvate kinase of bacterial community seemed to play important roles in stimulating the lactic acid fermentation, and the promotion of arginine deiminase could help lactic acid bacteria to tolerate the acidic environment. Conclusion: High-throughput sequencing technology combined with 16S rRNA gene-predicted functional analyses revealed the differences during the early and late stages of Sudangrass ensiling not only for distinct bacterial community but also for specific functional metabolites. The results could provide a comprehensive insight into bacterial community and metabolic characteristics to further improve the silage quality.

• Molecules and Cells
• /
• 제45권8호
• /
• pp.550-563
• /
• 2022

Hepatocellular carcinoma (HCC) is an aggressive and incurable cancer. Although understanding of the molecular pathogenesis of HCC has greatly advanced, therapeutic options for the disease remain limited. In this study, we demonstrated that SETD5 expression is positively associated with poor prognosis of HCC and that SETD5 depletion decreased HCC cell proliferation and invasion while inducing cell death. Transcriptome analysis revealed that SETD5 loss downregulated the interferon-mediated inflammatory response in HCC cells. In addition, SETD5 depletion downregulated the expression of a critical glycolysis gene, PKM (pyruvate kinase M1/2), and decreased glycolysis activity in HCC cells. Finally, SETD5 knockdown inhibited tumor growth in xenograft mouse models. These results collectively suggest that SETD5 is involved in the tumorigenic features of HCC cells and that targeting SETD5 may suppress HCC progression.

• Korean Journal of Acupuncture
• /
• 제39권4호
• /
• pp.142-151
• /
• 2022

Objectives : Parkinson's disease (PD) is a neurodegenerative disorder threatening the quality of life and highly occurred in over 65 years old. Gastrodia elata Blume (GEB), a traditional medicine used for the treatment of headache and convulsion, has been reported to have neuroprotective effect. This study was designed to investigate the neuroprotective effect of GEB and the proteomic changes in the substantia nigra (SN) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Methods : Male eleven-week-old C57BL/6 mice were intraperitoneally injected with 30 mg/kg of MPTP at 24-h intervals for 5 days. Two hours after the daily MPTP injection, the mice were orally administered 800 mg/kg of GEB extract, which continued for 7 days beyond the MPTP injections, for a total of 12 consecutive days. Two hours after the final GEB administration, the brain samples were collected, and dopaminergic neuronal death and proteomic changes in the SN were evaluated. Results : GEB prevented the MPTP-induced dopaminergic neuronal death and regulated the expression of 11 proteins including thimet oligopeptidase, T-complex protein 1, glycine tRNA ligase, and pyruvate kinase isozymes M1. Conclusions : GEB prevents MPTP-induced dopaminergic neuronal death by regulating the proteins in the SN.

• Mycobiology
• /
• 제50권5호
• /
• pp.366-373
• /
• 2022

Regulation of proper gene expression is important for cellular and organismal survival, maintenance, and growth. Abnormal gene expression, even for a single critical gene, can thwart cellular integrity and normal physiology to cause diseases, aging, and death. Therefore, gene expression profiling serves as a powerful tool to understand the pathology of diseases and to cure them. In this study, the difference in gene expression in Flammulina velutipes was compared between the wild type (WT) mushroom and the mutant one with clogging phenomenon. Differentially expressed transcripts were screened to identify the candidate genes responsible for the mutant phenotype using the DNA microarray analysis. A total of 88 genes including 60 upregulated and 28 downregulated genes were validated using the real-time quantitative PCR analysis. In addition, proteomic differences between the WT and mutant mushroom were analyzed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Interestingly, the genes identified by these genomic and proteomic analyses were involved in stress response, translation, and energy/sugar metabolism, including HSP70, elongation factor 2, and pyruvate kinase. Together, our data suggest that the aberrant expression of these genes attributes to the mutant clogging phenotype. We propose that these genes can be targeted to foster normal growth in F. velutipes.

• Journal of Ginseng Research
• /
• 제41권3호
• /
• pp.257-267
• /
• 2017

Background: Heat-processed ginseng, sun ginseng (SG), has been reported to have improved therapeutic properties compared with raw forms, such as increased antidiabetic, anti-inflammatory, and antihyperglycemic effects. The aim of this study was to investigate the antiobesity effects of SG through the suppression of cell differentiation and proliferation of mouse 3T3-L1 preadipocyte cells and the lipid accumulation in Caenorhabditis elegans. Methods: To investigate the effect of SG on adipocyte differentiation, levels of stained intracellular lipid droplets were quantified by measuring the oil red O signal in the lipid extracts of cells on differentiation Day 7. To study the effect of SG on fat accumulation in C. elegans, L4 stage worms were cultured on an Escherichia coli OP50 diet supplemented with $10{\mu}g/mL$ of SG, followed by Nile red staining. To determine the effect of SG on gene expression of lipid and glucose metabolism-regulation molecules, messenger RNA (mRNA) levels of genes were analyzed by real-time reverse transcription-polymerase chain reaction analysis. In addition, the phosphorylation of Akt was examined by Western blotting. Results: SG suppressed the differentiation of 3T3-L1 cells stimulated by a mixture of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI), and inhibited the proliferation of adipocytes during differentiation. Treatment of C. elegans with SG showed reductions in lipid accumulation by Nile red staining, thus directly demonstrating an antiobesity effect for SG. Furthermore, SG treatment down-regulated mRNA and protein expression levels of peroxisome proliferator-activated receptor subtype ${\gamma}$ ($PPAR{\gamma}$) and CCAAT/enhancer-binding protein-alpha ($C/EBP{\alpha}$) and decreased the mRNA level of sterol regulatory element-binding protein 1c in MDI-treated adipocytes in a dose-dependent manner. In differentiated 3T3-L1 cells, mRNA expression levels of lipid metabolism-regulating factors, such as amplifying mouse fatty acid-binding protein 2, leptin, lipoprotein lipase, fatty acid transporter protein 1, fatty acid synthase, and 3-hydroxy-3-methylglutaryl coenzyme A reductase, were increased, whereas that of the lipolytic enzyme carnitine palmitoyltransferase-1 was decreased. Our data demonstrate that SG inversely regulated the expression of these genes in differentiated adipocytes. SG induced increases in the mRNA expression of glycolytic enzymes such as glucokinase and pyruvate kinase, and a decrease in the mRNA level of the glycogenic enzyme phosphoenol pyruvate carboxylase. In addition, mRNA levels of the glucose transporters GLUT1, GLUT4, and insulin receptor substrate-1 were elevated by MDI stimulation, whereas SG dose-dependently inhibited the expression of these genes in differentiated adipocytes. SG also inhibited the phosphorylation of Akt (Ser473) at an early phase of MDI stimulation. Intracellular nitric oxide (NO) production and endothelial nitric oxide synthase mRNA levels were markedly decreased by MDI stimulation and recovered by SG treatment of adipocytes. Conclusion: Our results suggest that SG effectively inhibits adipocyte proliferation and differentiation through the downregulation of $PPAR{\gamma}$ and $C/EBP{\alpha}$, by suppressing Akt (Ser473) phosphorylation and enhancing NO production. These results provide strong evidence to support the development of SG for antiobesity treatment.

• Asian-Australasian Journal of Animal Sciences
• /
• 제28권11호
• /
• pp.1649-1656
• /
• 2015

Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect $A_{260}/A_{280}$ and $A_{260}/A_{230}$ ($p{\geq}0.05$), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples ($p{\geq}0.05$), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing.

• 한국미생물·생명공학회지
• /
• 제35권4호
• /
• pp.272-277
• /
• 2007

P. pastoris는 대장균에 비해 정확한 접힘, 당화, 효율적인 분비기작등의 장점을 가지고 있어 재조합 단백질의 생산을 위한 균주로서 관심을 받고 있다. 또한 재조합 단백질의 효율적인 생산공정 개발을 위하여 배양공정 중 특정 시간이나 조건에서 발현될 수 있는 효율적인 유도성 프로모터의 개발도 중요 관심 분야이다. 본 연구에서는 연속배양을 이용하여 P. pastoris의 배양 중 특정 기질이 소모되었을 때 발현되는 자동 유도성 프로모터를 개발하기 위하여, 인산이 고갈되었을 때 과발현 되는 유전자들을 탐색하였다. 인산 제한 연속배양의 정상상태에서 얻어진 균체로 부터 total RNA와 mRNA를 분리하였고, 이로부터 cDN를 합성하여 인산제한조건에서 과발현되는 유전자들을 확보하였다. 그 중 빈도수가 높은 8종의 유전자 3-phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase(GAPDH), glucokinase, thiol-specific antioxidant protein, triosephosphate isomerase, sodium/phosphate symporter(NPS) 그리고 pyruvate decarboxylase를 선별하였고, Northern blot analysis를 수행한 결과 인산 섭취에 관련된 NPS 유전자가 인산제한조건에서 과발현 됨을 확인하였다. 본 연구실에서는 자동유도성 프로모터로서 NPS유래의 프로모터의 잠재성을 알아보기 위하여, 관련 유전자를 확보하여 외래 유전자를 이용한 발현연구를 진행 중이다.