• Journal of Genetic Medicine
• /
• 제12권1호
• /
• pp.25-30
• /
• 2015

Purpose: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. Materials and Methods: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. Results: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. Conclusion: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

• Journal of Pharmaceutical Investigation
• /
• 제30권3호
• /
• pp.191-199
• /
• 2000

Genomics is providing targets faster than we can validate them and combinatorial chemistry is providing new chemical entities faster than we can screen them. Historically, the drug discovery cascade has been established as a sequential process initiated with a potency screening against a selected biological target. In this sequential process, pharmacokinetics was often regarded as a low-throughput activity. Typically, limited pharmacokinetics studies would be conducted prior to acceptance of a compound for safety evaluation and, as a result, compounds often failed to reach a clinical testing due to unfavorable pharmacokinetic characteristics. A new paradigm in drug discovery has emerged in which the entire sample collection is rapidly screened using robotized high-throughput assays at the outset of the program. Higher-throughput pharmacokinetics (HTPK) is being achieved through introduction of new techniques, including automation for sample preparation and new experimental approaches. A number of in vitro and in vivo methods are being developed for the HTPK. In vitro studies, in which many cell lines are used to screen absorption and metabolism, are generally faster than in vivo screening, and, in this sense, in vitro screening is often considered as a real HTPK. Despite the elegance of the in vitro models, however, in vivo screenings are always essential for the final confirmation. Among these in vivo methods, cassette dosing technique, is believed the methods that is applicable in the screening of pharmacokinetics of many compounds at a time. The widespread use of liquid chromatography (LC) interfaced to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) allowed the feasibility of the cassette dosing technique. Another approach to increase the throughput of in vivo screening of pharmacokinetics is to reduce the number of sample analysis. Two common approaches are used for this purpose. First, samples from identical study designs but that contain different drug candidate can be pooled to produce single set of samples, thus, reducing sample to be analyzed. Second, for a single test compound, serial plasma samples can be pooled to produce a single composite sample for analysis. In this review, we validated the issue whether the second method can be applied to practical screening of in vivo pharmacokinetics using data from seven of our previous bioequivalence studies. For a given drug, equally spaced serial plasma samples were pooled to achieve a 'Pooled Concentration' for the drug. An area under the plasma drug concentration-time curve (AUC) was then calculated theoretically using the pooled concentration and the predicted AUC value was statistically compared with the traditionally calculated AUC value. The comparison revealed that the sample pooling method generated reasonably accurate AUC values when compared with those obtained by the traditional approach. It is especially noteworthy that the accuracy was obtained by the analysis of only one sample instead of analyses of a number of samples that necessitates a significant man-power and time. Thus, we propose the sample pooling method as an alternative to in vivo pharmacokinetic approach in the selection potential lead(s) from combinatorial libraries.

• Journal of Animal Science and Technology
• /
• 제61권5호
• /
• pp.245-253
• /
• 2019

Pathogen-associated Molecular Patterns (PAMPs) are highly conserved structural motifs that are recognized by Pathogen Recognition receptors (PRRs) to initiate immune responses. Infection by these pathogens and the immune response to PAMPS such as lipopolysaccharide (LPS), Peptidoglycan (PGN), bacterial oligodeoxynucleotides [CpG oligodeoxynucleotides 2006 (CpG ODN2006) and CpG oligodeoxynucleotides 2216 (CpG ODN2216)], and viral RNA Polyinosinic-Polycytidylic Acid (Poly I:C), are associated with infectious and metabolic diseases in animals impacting health and production. It is established that PAMPs mediate the production of cytokines by binding to PRRs such as Toll-like receptors (TLR) on immune cells. Galectins (Gal) are carbohydrate-binding proteins that when expressed play essential roles in the resolution of infectious and metabolic diseases. Thus it is important to determine if the expression of galectin gene (LGALS) and Gal secretion in blood are affected by exposure to LPS and PGN, PolyI:C and bacterial CpG ODNs. LPS increased transcription of LGALS4 and 12 (2.5 and 2.02 folds respectively) and decreased secretion of Gal 4 (p < 0.05). PGN increased transcription of LGALS-1, -2, -3, -4, -7, and -12 (3.0, 2.3, 2.0, 4.1, 3.3, and 2.4 folds respectively) and secretion of Gal-8 and Gal-9 (p < 0.05). Poly I:C tended to increase the transcription of LGALS1, LGALS4, and LGALS8 (1.78, 1.88, and 1.73 folds respectively). Secretion of Gal-1, -3, -8 and nine were significantly increased in treated samples compared to control (p < 0.05). CpG ODN2006 did not cause any significant fold changes in LGALS transcription (FC < 2) but increased secretion of Gal-1, and-3 (p < 0.05) in plasma compared to control. Gal-4 was however reduced in plasma (p < 0.05). CpG ODN2216 increased transcription of LGALS1 and LGALS3 (3.8 and 1.6 folds respectively), but reduced LGALS2, LGALS4, LGALS7, and LGALS12 (-1.9, -2.0, -2.0 and; -2.7 folds respectively). Secretion of Gal-2 and -3 in plasma was increased compared to control (p < 0.05). Gal-4 secretion was reduced in plasma (p < 0.05). The results demonstrate that PAMPs differentially modulate galectin transcription and translation of galectins in cow blood.

• 한국해양바이오학회지
• /
• 제14권1호
• /
• pp.1-8
• /
• 2022

This study examined the growth, fatty acid (FA) content, and carotenoids of a newly isolated freshwater microalga, Mychonastes sp. 246, in various culture media. The appropriate temperature and light intensity for culturing Mychonastes sp. 246 were determined as 18℃-22℃ and 200-250 µmol/m²/s using a high throughput photobioreactor. The microalgal cells were cultivated in 0.5 L bubble column photobioreactors using BG11, Bold's Basal media, and f/2 media. According to the growth results of the microalgae, BG11, among the tested media, showed the highest biomass concentrations (3.5 ± 0.1 g/L in 10 d). To enhance the biomass growth of the microalgae, the N:P ratio in BG11 was manipulated from 45:1 to 7:1 based on the stoichiometric cell composition. The biomass concentrations of Mychonastes sp. 246 grown on the manipulated BG11 (MBG) increased to 38% (4.6 ± 0.3 g/L in d) compared with the original BG11 (3.3 g/L). The FA content of the microalgae grown on the MBG was lower (8.4%) than that of the original BG11 (10.1%) while the FA compositions did not exhibit any significant differences. Furthermore, three kinds of carotenoids were identified in Mychonastes sp. 246, zeaxanthin, lutein, and β-carotene. These results suggest an effective strategy for increasing biomass concentrations, FA content, and carotenoids of microalgae by performing a simple N:P adjustment in the culture media.

• 한국가금학회지
• /
• 제38권4호
• /
• pp.293-304
• /
• 2011

본 시험은 한국토종닭의 3원 교배가 생산성과 육질에 미치는 영향을 조사하기 위해 수행되었다. 공시계는 국립축산과학원 축산자원개발부에서 보유하고 있는 한국토종닭 순종을 3원 교배하여 발생한 암수 병아리 540수와 백세미 180수를 이용하였다. 시험 설계는 A) CS${\times}$B, B) CH${\times}$S, C) RS${\times}$H, D) 백세미에서 발생된 4 교배종의 병아리를 각각 암수 구분하여, $4{\times}2$의 총 8교배종, 교배종당 9반복, 반복당 10수씩($4{\times}2{\times}9{\times}10$) 총 720수를 완전임의 배치하였다. 목표 체중에 도달했을 때(5주령과 10주령), 교배 조합에 따라 각각 수컷 9수씩 도축하여 도체율과 부분육(날개, 등, 목, 가슴, 다리) 비율을 조사하고 육질검사를 하였다. 수정율은 처리구간에 차이가 없었으며, 부화율은 B 교배종이 가장 낮게 나타났다. 체중은 5주령에 수컷의 체중이 높게 나타났으며(P<0.05), 증체량, 사료 요구율은 A 교배종이 가장 높았다(P<0.05). 도체율의 경우에는, 5주령에 A 교배종이 가장 높았으나(P<0.05), 10주령에는 교배종간 차이가 없었다. 부분육의 경우, 5주령시에는 C 교배종의 날개, 목, 가슴, 다리의 비율이 다른 교배종에 비해 낮았으나(P<0.05), 가슴 부위는 다른 교배종과 차이가 없었다(P>0.05). 10주령에는 A 교배종의 가슴 비율이 가장 높았으며(P<0.05), 다른 부분육의 비율은 교배종간 차이가 없었다(P<0.05). 5주령 계육의 화학적 성상을 보면, pH는 교배 조합 사이에서 유의차가 없었으며, 수분의 함량은 D 교배종, 단백질 함량은 B 교배종이 가장 높았다(P<0.05). 지방과 회분 함량은 교배 조합 사이에서 유의차가 없었다. 10주령의 계육의 화학적 성상은 pH가 A 교배 조합에서 가장 높았고, 수분, 지방, 단백질 및 회분 함량은 교배 조합 사이에서 차이가 없었다(P>0.05). 5주령의 육색은 명도($L^*$)와 적색도($a^*$)는 교배종간 차이가 없었으나, 10주령의 육색은 A 교배종의 적색도($a^*$)가 가장 높았다(P<0.05). 5주령에서 전단력과 가열 감량은 A 교배종이 높게 나타났으나(P<0.05). 10주령의 가열 감량은 A 교배종이 다른 교배종들에 비해 낮았다(P<0.05).