• Asian-Australasian Journal of Animal Sciences
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• v.25 no.12
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• pp.1681-1690
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• 2012

The aim of this study was to investigate the impact of a reported p53 inhibitor, pifithrin-${\alpha}$ (PFT-${\alpha}$), on preimplantation porcine in vitro fertilized (IVF) embryo development in culture. Treatment of PFT-${\alpha}$ was administered at both early (0 to 48 hpi), and later stages (48 to 168 hpi) of preimplantation development, and its impact upon the expression of five genes related to apoptosis (p53, bak, bcl-xL, p66Shc and caspase3), was assessed in resulting d 7 blastocysts, using real-time quantitative PCR. Total cell numbers, along with the number of apoptotic nuclei, as detected by the in situ cell death detection assay, were also calculated on d 7 in treated and non-treated control embryos. The results indicate that PFT-${\alpha}$, when administered at both early and later stages of porcine IVF embryo development, increases the incidence of apoptosis in resulting blastocysts. When administered at early cleavage stages, PFT-${\alpha}$ treatment was shown to reduce the developmental competence of porcine IVF embryos, as well as reducing the quality of resulting blastocysts in terms of overall cell numbers. In contrast, at later stages, PFT-${\alpha}$ administration resulted in marginally increased blastocyst development rates amongst treated embryos, but did not affect cell numbers. However, PFT-${\alpha}$ treatment induced apoptosis and apoptotic related gene expression, in all treated embryos, irrespective of the timing of treatment. Our results indicate that PFT-${\alpha}$ may severely compromise the developmental potential of porcine IVF embryos, and is a potent apoptotic agent when placed into porcine embryo culture media. Thus, caution should be exercised when using PFT-${\alpha}$ as a specific inhibitor of p53 mediated apoptosis, in the context of porcine IVF embryo culture systems.

• Animal Bioscience
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• v.34 no.12
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• pp.1879-1885
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• 2021

Objective: Owing to the lack of a breeding index for efficient and quick fertility evaluations of Holstein bulls when using traditional or genome-wide detection methods, this study aimed to determine whether in vitro fertilization (IVF) could be used as an indicator of conception rate of artificial insemination (AI). Methods: Conventional and sexed frozen semen from nine bulls were used for IVF and AI. Results: The IVF and AI conception rates of each bull were confirmed to be positively correlated between the conventional frozen and sexed frozen semen. The correlation coefficient R values of nine bulls between IVF and AI methods were 0.73 and 0.97 for the conventional frozen and sexed frozen semen, respectively. The average conception rate of three bulls undergoing AI was 69.5% and 64.2%, 61.8% and 58.8%, and 48.2% and 46.2% in first-, second-, and third-born cows when conventional frozen and sexed frozen semen were used, respectively, which showed a positive correlation with the fertilization rate in the same parity. We propose an evaluation standard to assess the fertilization ability of bulls based on their IVF test results, which is categorized into three grades: grade one, normal fertility bull with an AI conception rate of 40%±5% and IVF rate of 45% to 60%; grade two, higher fertility bull with an AI conception rate of 50%±5% and IVF rate of 61% to 80%; and grade three, highest fertility bull with an AI conception rate of 60%±5% and IVF rate of >80%. Conclusion: These findings reveal that IVF results can be used as a breeding index for bulls to evaluate their AI conception ability, which may shorten the time required to select bulls for breeding.

• Journal of Microbiology and Biotechnology
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• v.26 no.3
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• pp.588-595
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• 2016

IFN-γ release assays (IGRAs) have been developed as viable alternative diagnostic tools for detecting latent tuberculosis infection (LTBI). A customized homogeneous sandwich luminescent oxygen channeling immunoassay (LOCI) was used to quantify IFN-γ levels in IGRAs. Samples were collected from healthy volunteers (n = 40) who were T-Spot-negative and T-Spot-positive patients (n = 32) at rest. Then the amount of IFN-γ in the supernatant of IGRAs was measured by LOCI. The results demonstrated a low background, and high sensitivity, specificity, accuracy, and reproducibility, and a short assay time (only 30 min) with LOCI for IFN-γ. The recovery range was 81.63-102.06%, the coefficients of variation were below 5%, and the limit of detection was 19.0 mIU/ml. Excellent agreement between LOCI IFN-γ and the T-SPOT.TB test was obtained (97.2% agreement, κ = 0.94). The LOCI IFN-γ concentrations were significantly higher in T-Spot-positive patients than in the healthy group (p < 0.001). Moreover, as observed for the comparative LOCI IFN-γ assay, IFN-γ concentrations were related to the numbers of T-SPOT.TB spots. We have established an in vitro blood test for LTBI diagnosis, defined as LOCI IFN-γ. A high level of agreement between the LOCI IFN-γ method and T-SPOT.TB assay was observed in clinical studies that showed the LOCI IFN-γ method could determine LTBI. This study shows acceptable performance characteristics of the LOCI IFN-γ assay to diagnose LTBI.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.2 no.2
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• pp.96-102
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• 2016

Apoptosis, a genetically determined process of programmed cell death, is considered a vital component of various processes including normal cell turnover, animal development, and tissue homeostasis. It has a crucial role in many medical disorders and hence the development of non-invasive imaging tool is highly demanded. Recently, we have developed a peptide-based radioactive probe (ApoPep-1) for apoptosis detection. In that work the potential of probe for apoptosis detection was verified, however in vivo stability of radiolabeled peptide was not enough to monitor apoptosis for extended period. In current study, we prepared cyclic ApoPep-1 peptides to improve the stability of origianl linear ApoPep-1 and carried out direct comparison studies in vitro and in vivo. A targeting efficacy of newly synthesized cyclic ApoPep-1 peptide for apoptosis was confirmed in acute myocardial infarct model.

• Archives of Pharmacal Research
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• v.9 no.2
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• pp.99-110
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• 1986

In order to use for metabolic studies of tranylcypromine (TCP), TCP-phenyl-$d_{5}$ was synthesized via the intermediates, 3-benzoylpropionic acid-$d_{5}$ and trans-2-phenylcyclopropanecarboxylic acid-$d_{5}$ -TCP(0.22 mmole/kg) and its deuterated analog were administered s. c. to the rats and GC/MS analyses of the urines led to the detection of N-acetyltranylcypromine (ATCP) and glucuronide conjugate of phenyl-hydroxylated ATCP. MAO activities in rat brain were measured using serotonin as the substrate. In vitro $IC_{50}$ of ATCP was determined to be $10^{-3}M$. The inhibitions by ATCP were not dependent on the preincubation time and were reversed by washing sedimented mitochondrial pellets after the preincubation. In vivo MAO inhibitions at various times of 0.5, 1.5, 3, 6, 12, and 23 hr after the administration of 0.4 mmole/kg (i. p. ) of ATCP were found to be 0.13, 73, 90, 89, and 74 %, respectively. Similarly, the inhibition percents by 0.015 mmole/kg (i. p. ) of TCP were 94, 99, 95, 91, 71 and 49%. The results strongly suggest that deacetylated product of ATCP may account for its in vivo MAO inhibition. The relationship between the metabolism via phenyl-hydroxylation and the in vivo potency of TCP was examined by QSAR study and it was found that groupings discriminating between the compounds with p-substituents and those without them only ensure high correlations, suggesting that ring-hydroxylation which occurs at the para position in most of the compounds is a determining factor to the potency of TCP.

• Plant Disease and Agriculture
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• v.5 no.1
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• pp.27-33
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• 1999

Frost injury of crops is closely related to the epiphytic population dynamics of ice nucleation-active (INA) bacteria, and the injury can be reduced by decreasing the INA bacterial population. In order to predict the epiphytic population of INA bacteria on crops, a rapid and accurate detection method has to be developed. In the previous report, we produced some antibodies against INA proteins purified from the outer membrane of INA bacteria. However it was difficult to produce the antibodies because the purification procedures of the INA proteins were complicated, and the final yield was too low. We designed a specific peptide from the N-terminal region of INA protein by computer analysis and synthesized the peptide in vitro in this experiment. The peptide sequence was Asp-Ser-Por-Leu-Ser-Leu-His-Ala-Asp, that is corresponding to the highly conserved region in several INA proteins, with predicted beta turn, coiling, and hydrophilic region. A polyclonal anti-INA peptide antiserum produced specifically recognized INA bacteria as few as 10 colony-forming units (CFU) in the ELISA reactions and did not respond to other non-INA bacteria. Serological specificity of the anti-INA peptide antiserum will facilitate the forecasting of the INA bacterial population dynamics on crops.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.145-152
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• 2008

A cell-based in vitro exposure system was developed to determine whether oxidative stress plays a role in the cytotoxic effects of volatile organic compounds (VOCs) such as benzene, toluene, xylene, and chlorobenzene, using human epithelial HeLa cells. Thin films based on cysteine-terminated synthetic oligopeptides were fabricated for immobilization of the HeLa cells on a gold (Au) substrate. In addition, an immobilized cell-based sensor was applied to the electrochemical detection of the VOCs. Layer formation and immobilization of the cells were investigated with surface plasmon resonance (SPR), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The adhered living cells were exposed to VOCs; this caused a change in the SPR angle and the VOC-specific electrochemical signal. In addition, VOC toxicity was found to correlate with the degree of nitric oxide (NO) generation and EIS. The primary reason for the marked increase in impedance was the change of aqueous electrolyte composition as a result of cell responses. The p53 and NF-${\kappa}B $ downregulation were closely related to the magnitude of growth inhibition associated with increasing concentrations of each VOC. Therefore, the proposed cell immobilization method, using a self-assembly technique and VOC-specific electrochemical signals, can be applied to construct a cell microarray for onsite VOC monitoring.

• Biomedical Science Letters
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• v.17 no.3
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• pp.191-196
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• 2011

Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least $10^6$ viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup I (GI) and genogroup II (GII) using standard viral RNA. For real-time NASBA assay which can detected noroV GI and GII, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV GI and GII. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV GI and GII were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.

• Journal of Plant Biotechnology
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• v.39 no.3
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• pp.196-204
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• 2012

This study was carried out to develop reliable systems for somatic embryogenesis in oil palm tree (Elaeis guineensis Jacq.), and to verify the somaclonal variants by RAPD analysis. Embryogenic callus was induced successfully on modified half-strength MS medium containing $NaH_2PO_4{\cdot}2H_2O$ and casein. Embryogenic callus was further developed to somatic embryo mass (SEM), which is very hard and bonded tightly each other. Plantlets were proliferated when SEM was cultured on modified MS medium containing half strength $NH_4NO_3$, casein and L-ascorbic acid. Plantlets were transplanted into pots containing artificial soils. When RAPD analysis was conducted using randomly selected 95 in vitro plantlets and 19 random primers, somaclonal variation was detected using BNR35 primer. There was missing band around 1 kb in #22, #28, #35, and #77 plantlets. In addition, bands obtained from #28, #35, and #77 was much stronger than other normal bands. The blast results at NCBI revealed that somaclonal variation observed in this study was related to chloroplast genome of oil palm. The results also revealed that oil palm reproduction system through somatic embryogenesis is quite reliable and early detection of somaclonal variants seem to be possible at in vitro stage by RAPD analysis.

• Journal of Life Science
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• v.29 no.9
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• pp.1023-1026
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• 2019

The purpose of this study was to investigate the estrogen-like activities of eight oriental medicines, including red ginseng, fenugreek, and dandelion. Hot water and ethanol extracts were prepared from Cervus nippon temminck (Nokgaksang), Cynanchum wilfordii (Baeksuo), Lespedeza cuneata (Yagwanmun), Panax ginseng (red ginseng), Smilax china (Toryeong), Taraxacum platycarpum (Mindeulre, dandelion), Tribulus terresteis (Namgase), and Trigonella foenum-graecum (Horopa, fenugreek). Then, estrogen-like activities were verified by the in vitro transcriptional activity assay. The extracts showing estrogen-like activities were red ginseng, Baeksuo, fenugreek, Yagwanmun, and dandelion in the ethanol extracts, and red ginseng, fenugreek, and Baeksuo in the hydrothermal extracts. Red ginseng extract showed a higher activity than the standard $10^{-8}M$ $17{\beta}$-estradiol in both the hydrothermal and ethanol extract at $500{\mu}g/ml$, while the $50{\mu}g/ml$ of the red ginseng ethanol extract and the $500{\mu}g/ml$ of the Baeksuo hydrothermal extract showed estradiol activities between $10^{-9}$ and $10^{-8}M$ $17{\beta}$-estradiol. This shows that it would be possible to contribute to the development of functional materials using red ginseng, Baeksuo, fenugreek, and dandelion.