• 제목/요약/키워드: In vitro assays

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Ginsenoside 20(S)-Rg3 reduces KIF20A expression and promotes CDC25A proteasomal degradation in epithelial ovarian cancer

  • Rong Zhang;Lei Li;Huihui Li;Hansong Bai;Yuping Suo;Ju Cui;Yingmei Wang
    • Journal of Ginseng Research
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    • 제48권1호
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    • pp.40-51
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    • 2024
  • Background: Ginsenoside 20(S)-Rg3 shows promising tumor-suppressive effects in ovarian cancer via inhibiting NF-kB signaling. This study aimed to explore the downstream tumor suppressive mechanisms of ginsenoside Rg3 via this signaling pathway. Materials and methods: A systematical screening was applied to examine the expression profile of 41 kinesin family member genes in ovarian cancer. The regulatory effect of ginsenoside Rg3 on KIF20A expression was studied. In addition, we explored interacting proteins of KIF20A and their molecular regulations in ovarian cancer. RNA-seq data from The Cancer Genome Atlas (TCGA) was used for bioinformatic analysis. Epithelial ovarian cancer cell lines SKOV3 and A2780 were used as in vitro and in vivo cell models. Commercial human ovarian cancer tissue arrays were used for immunohistochemistry staining. Results: KIF20A is a biomarker of poor prognosis among the kinesin genes. It promotes ovarian cancer cell growth in vitro and in vivo. Ginsenoside Rg3 can suppress the transcription of KIF20A. GST pull-down and co-immunoprecipitation (IP) assays confirmed that KIF20A physically interacts with BTRC (β-TrCP1), a substrate recognition subunit for SCFβ-TrCP E3 ubiquitin ligase. In vitro ubiquitination and cycloheximide (CHX) chase assays showed that via interacting with BTRC, KIF20A reduces BTRC-mediated CDC25A poly-ubiquitination and enhances its stability. Ginsenoside Rg3 treatment partly abrogates KIF20A overexpression-induced CDC25A upregulation. Conclusion: This study revealed a novel anti-tumor mechanism of ginsenoside Rg3. It can inhibit KIF20A transcription and promote CDC25A proteasomal degradation in epithelial ovarian cancer.

Phototoxicity: Its Mechanism and Animal Alternative Test Methods

  • Kim, Kyuri;Park, Hyeonji;Lim, Kyung-Min
    • Toxicological Research
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    • 제31권2호
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    • pp.97-104
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    • 2015
  • The skin exposure to solar irradiation and photoreactive xenobiotics may produce abnormal skin reaction, phototoxicity. Phototoxicity is an acute light-induced response, which occurs when photoreacive chemicals are activated by solar lights and transformed into products cytotoxic against the skin cells. Multifarious symptoms of phototoxicity are identified, skin irritation, erythema, pruritis, and edema that are similar to those of the exaggerated sunburn. Diverse organic chemicals, especially drugs, are known to induce phototoxicity, which is probably from the common possession of UV-absorbing benzene or heterocyclic rings in their molecular structures. Both UVB (290~320 nm) and UVA (320~400 nm) are responsible for the manifestation of phototoxicity. Absorption of photons and absorbed energy (hv) by photoactive chemicals results in molecular changes or generates reactive oxygen species and depending on the way how endogenous molecules are affected by phototoxicants, mechanisms of phototoxcity is categorized into two modes of action: Direct when unstable species from excited state directly react with the endogenous molecules, and indirect when endogeneous molecules react with secondary photoproducts. In order to identify phototoxic potential of a chemical, various test methods have been introduced. Focus is given to animal alternative test methods, i.e., in vitro, and in chemico assays as well as in vivo. 3T3 neutral red uptake assay, erythrocyte photohemolysis test, and phototoxicity test using human 3-dimensional (3D) epidermis model are examples of in vitro assays. In chemico methods evaluate the generation of reactive oxygen species or DNA strand break activity employing plasmid for chemicals, or drugs with phototoxic potential.

P25: A hidden target for AD therapeutic.

  • Ha, Il-Ho
    • 한국응용약물학회:학술대회논문집
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    • 한국응용약물학회 2006년도 Spring Conference
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    • pp.95-105
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    • 2006
  • Alzheimer's disease (AD) is an irreversible, progressive brain disorder that is characterized by dementia. Amounts of p25 and cdk5 kinase activity are specifically upregulated in AD patient's brain samples. Considerable evidence now points importance of p25/cdk5 in generation of A$\beta$ peptides and hyperphosphorylation of tau linking amyloid plaques and neurofibrillary tangles, two major pathological hallmarks of AD. We demonstrated that P25/CDK5 phosphorylates BACE1, the first step protease to produce A$\beta$. P25/CDK5 inhibitors to reduce BACE1 phosphorylation and the secretion of A$\beta$ are screened through in silica, in vitro, and cell-based assays. Out of 4.3 million chemicals we finally selected two compounds to have IC50 of 10 microM in cell-based assays. The inhibitors block Tau phosphrorylation as well as BACE1 phosphorylation. In conclusion P25 should be one of the best targets for AD therapeutics.

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Toxicogenomics and Cell-based Assays for Toxicology

  • Tong, Weida;Fang, Hong;Mendrick, Donna
    • Interdisciplinary Bio Central
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    • 제1권3호
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    • pp.10.1-10.5
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    • 2009
  • Toxicity is usually investigated using a set of standardized animal-based studies which, unfortunately, fail to detect all compounds that induce human adverse events and do not provide detailed mechanistic information of observed toxicity. As an alternative to conventional toxicology, toxicogenomics takes advantage of currently advanced technologies in genomics, proteomics, metabolomics, and bioinformatics to gain a molecular level understanding of toxicity and to enhance the predictive power of toxicity testing in drug development and risk/safety assessment. In addition, there has been a renewed interest, particularly in various government agencies, to prioritize and/or supplement animal testing with a battery of mechanistically informative in vitro assays. This article provides a brief summary of the issues, challenges and lessons learned in these fields and discuss the ways forward to further advance toxicology using these technologies.

Use of in vitro assays for evaluating physiological functionality of foods: General consideration

  • Chun Hyang Sook
    • 한국식품영양과학회:학술대회논문집
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    • 한국식품영양과학회 2003년도 정기총회 및 54차 학술발표회:건강기능식품의 유효성평가
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    • pp.74-77
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    • 2003
  • A new health paradigm may be evolving that would place emphasis on the positive aspects of diet, identifying components that are physiologically active and that contribute to the prevention of disease onset. It has been increasingly difficult to evaluate the impact of new bioactive materials and food products on the well being of society. Thus, testing systems for both health function and toxicity have become very elaborate, complex, and interrelated, making their interpretation difficult and open to controversy. To select the proper starting materials, to screen the appropriate health functionality and to determine the efficacy of product, a reliable, reproducible, sensitive and predictive assay is required. Particularly, in uitro assay is the first stage in preclinical test on physiologically active materials and have many advantages in terms of time, cost and convenience. However, several factors as well as some limitations should be considered in this assay system. This presentation, therefore, will address the use of in uitro assays for evaluating physiological functionality of foods coupled with general consideration.

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살충제 이미다크로프리드 잔류물의 실시간 측정용 효소면역분석법 (Enzyme Immunoassay for On-line Sensing of the Insecticide Imidaclopird Residues)

  • 송석진;조한근
    • Journal of Biosystems Engineering
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    • 제28권6호
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    • pp.505-510
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    • 2003
  • In Korea, due to its broad efficacy as a systemic insecticide, imidacloprid has been widely used in rice paddies to control sucking insects, soil insects, and some chewing insects and in apple orchards to control various insects pests. To quantify the imidacloprid residue concentrations, samples are assayed in vitro using enzyme-linked immunosorbent assays(ELISA). These assays generally require several hours to perform. As a biosensor, a competitive imidacloprid ELISA was modified to measure insecticide concentrations. It was found that a total assay time of 15 min(10-min antibody-antigen binding, and 5-min substrate development) is sufficient for monitoring imidacloprid concentrations. Further work is needed to improve the sensitivity of the measurement protocol.

In vitro Antifungal Activity of Limonene against Trichophyton rubrum

  • Chee, Hee-Youn;Kim, Hoon;Lee, Min-Hee
    • Mycobiology
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    • 제37권3호
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    • pp.243-246
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    • 2009
  • In this study, the antifungal activities of limonene against Trichophyton rubrum were evaluated via broth microdilution and vapor contact assays. In both assays, limonene was shown to exert a potent antifungal effect against T. rubrum. The volatile vapor of limonene at concentrations above $1{\mu}l$/800 ml air space strongly inhibited the growth of T. rubrum. The MIC value was 0.5% v/v in the broth microdilution assay. The antifungal activity of limonene against T. rubrum was characterized as a fungicidal effect.

Biofilm-forming ability and adherence to poly-(methyl-methacrylate) acrylic resin materials of oral Candida albicans strains isolated from HIV positive subjects

  • Uzunoglu, Emel;Bicer, Arzu Zeynep Yildirim;Dolapci, Istar;Dogan, Arife
    • The Journal of Advanced Prosthodontics
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    • 제6권1호
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    • pp.30-34
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    • 2014
  • PURPOSE. This study evaluated the adhesion to acrylic resin specimens and biofilm formation capability of Candida albicans strains isolated from HIV positive subjects' oral rinse solutions. MATERIALS AND METHODS. The material tested was a heat-cured acrylic resin (Acron Duo). Using the adhesion and crystal violet assays, 14 oral Candida albicans isolated from HIV-positive subjects and 2 references Candida strains (C. albicans ATCC 90028 and C. albicans ATCC 90128) were compared for their biofilm production and adhesion properties to acrylic surfaces in vitro. RESULTS. There were no significant differences in adhesion (P=.52) and biofilm formation assays (P=.42) by statistical analysis with Mann-Whitney test. CONCLUSION. Denture stomatitis and increased prevalence of candidal carriage in HIV infected patients is unlikely to be related to the biofilm formation and adhesion abilities of C. albicans to acrylic resin materials.

In vitro cytotoxicity and in vivo acute toxicity of selected polysaccharide hydrogels as pharmaceutical excipients

  • Kulkarni GT;Gowthanarajan K;Raghu C;Ashok G;Vijayan P
    • Advances in Traditional Medicine
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    • 제5권1호
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    • pp.29-36
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    • 2005
  • Polysaccharide hydrogels constitute a structurally diverse class of biological macromolecules with a wide range of physicochemical properties. They also constitute important members of the family of industrial water-soluble polymers. They find application in Pharmacy as binders, disintegrants, suspending, emulsifying and sustaining agents. According to the International Pharmaceutical Excipients Council (IPEC), an excipient must have an established safety profile. Hence, in the present study, in vitro cytotoxicity on Vero and HEp-2 cell lines, and in vivo acute toxicity in rats were carried out to establish the safety of polysaccharide hydrogels from the seeds of Plantago ovata and Ocimum basilicum. The in vitro cytotoxicity was determined by MTT and SRB assays. In the in vivo acute toxicity, the effects of three different doses of hydrogels (100, 200 and 400 mg/kg body weight) on food and water intake, body weight, biochemical and hematological parameters were studied. The results of in vitro did not show any cytotoxicity on both the cell lines used. In the in vivo acute toxicity, the hydrogels did not show any toxic symptoms in all three dose levels. This establishes the safety of the selected hydrogels. Hence, they can be used as excipients in pharmaceutical dosage forms.

Nutrient digestibility in black soldier fly larva was greater than in adults for pigs and could be estimated using fiber

  • Jongkeon Kim;Kwanho Park;Sang Yun Ji;Beob Gyun Kim
    • Journal of Animal Science and Technology
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    • 제65권5호
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    • pp.1002-1013
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    • 2023
  • The objectives of the present study were to determine the nutrient digestibility of fish meal, defatted black soldier fly larvae (BSFL), and adult flies and to develop equations for estimating in vitro nutrient digestibility of BSFL for pigs. In vitro digestion procedures were employed to mimic the digestion and absorption of nutrients in the pig intestine. Correlation coefficients between chemical composition and in vitro nutrient digestibility of BSFL were calculated. In Exp. 1, in vitro ileal digestibility (IVID) of dry matter (DM) and crude protein (CP) and in vitro total tract digestibility (IVTTD) of DM and organic matter in defatted BSFL meal were less (p < 0.05) than those in fish meal but were greater (p < 0.05) than those in adult flies. In Exp. 2, CP concentrations in BSFL were negatively correlated with ether extract (r = -0.91) concentration but positively correlated with acid detergent fiber (ADF; r = 0.98) and chitin (r = 0.95) concentrations. ADF and chitin concentrations in BSFL were negatively correlated with IVID of DM (r = -0.98 and -0.88) and IVTTD of DM (r = -1.00 and -0.94) and organic matter (r = -0.99 and -0.98). Prediction equations for in vitro nutrient digestibility of BSFL were developed: IVID of CP (%) = -0.95 × ADF (% DM) + 95 (r2 = 0.75 and p = 0.058) and IVTTD of DM (%) = -2.09 × ADF + 113 (r2 = 0.99 and p < 0.001). The present in vitro experiments suggest that defatted BSFL meal was less digestible than fish meal but was more digestible than adult flies, and nutrient digestibility of BSFL can be predicted using ADF as an independent variable.