• Asian-Australasian Journal of Animal Sciences
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• 제11권4호
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• pp.428-433
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• 1998

The effects of chromium picolinate supplementation in pig diet were evaluated by measuring the in vitro lipogenic and lipolytic activities in adipose tissue and the protein synthetic activity in liver acinar cell in culture. Thirty-two male and thirty-two female pigs were randomly assigned to one of four dietary groups: Control, 100 ppb, 200 ppb, and 400 ppb of Cr in the form of picolinate. The chromium picolinate supplementation (p < 0.01) increased the in vitro lipolytic activity in adipose tissue of pig, but had no effects on lipogenesis. The chromium picolinate effect was greater in female pigs than in male pigs on lipolytic activity. The results from the studies with the liver acinar cells in culture indicated that chromium picolinate supplementation increased protein synthetic activity (p < 0.05). It was observed through this experiment that chromium picolinate functions not only on fat degradation but also on retained protein synthesis.

• Molecules and Cells
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• 제39권5호
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• pp.410-417
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• 2016

During the lactation cycle of the bovine mammary gland, autophagy is induced in bovine mammary epithelial cells (BMECs) as a cellular homeostasis and survival mechanism. Interferon gamma ($IFN-{\gamma}$) is an important antiproliferative and apoptogenic factor that has been shown to induce autophagy in multiple cell lines in vitro. However, it remains unclear whether $IFN-{\gamma}$ can induce autophagy and whether autophagy affects milk synthesis in BMECs. To understand whether $IFN-{\gamma}$ affects milk synthesis, we isolated and purified primary BMECs and investigated the effect of $IFN-{\gamma}$ on milk synthesis in primary BMECs in vitro. The results showed that $IFN-{\gamma}$ significantly inhibits milk synthesis and that autophagy was clearly induced in primary BMECs in vitro within 24 h. Interestingly, autophagy was observed following $IFN-{\gamma}$ treatment, and the inhibition of autophagy can improve milk protein and milk fat synthesis. Conversely, upregulation of autophagy decreased milk synthesis. Furthermore, mechanistic analysis confirmed that $IFN-{\gamma}$ mediated autophagy by depleting arginine and inhibiting the general control nonderepressible-2 kinase (GCN2)/eukaryotic initiation factor $2{\alpha}$ ($eIF2{\alpha}$) signaling pathway in BMECs. Then, it was found that arginine supplementation could attenuate $IFN-{\gamma}$-induced autophagy and recover milk synthesis to some extent. These findings may not only provide a novel measure for preventing the $IFN-{\gamma}$-induced decrease in milk quality but also a useful therapeutic approach for $IFN-{\gamma}$-associated breast diseases in other animals and humans.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.420-424
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• 2006

A simple method for depleting E. coliS30 extracts of endogenous tRNA has been developed. An $ethanolamine-Sepharose^{(R)}$ column equilibrated with water selectively captured the tRNA molecules in E. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful for in vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.

• 생명과학회지
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• 제15권5호
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• pp.779-782
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• 2005

Coupled transcription/translation cocktail을 이용하여 R. glutinis EH 유전자를 in vitro에서 합성하고 활성을 평가하였다. SDS-PAGE 및 immunoblotting을 통하여 45 kDa 크기의 EH 단백질이 발현되었음을 확인하였고, NBP assay 및 chiral GC 분석을 통해 발현된 단백질이 (R)-styrene oxide에 대한 입체선택성이 있음을 확인하였다. 따라서 무세포 단백질 합성 시스템을 이용하여 입체선택성을 유지시킨 EH 유전자 발현이 가능하며, 이러한 방법은 putative EH 유전자 탐색 등에 효율적으로 응용될 것이다.

• Asian-Australasian Journal of Animal Sciences
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• 제13권8호
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• pp.1084-1093
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• 2000

An in vitro gas production system was used to investigate the influence of various substrate mixtures on a natural mix of rumen microbes by measurement of fermentation end-products. The treatments were combinations of cassava (15.0, 30.0 and 45.0%) with different roughage sources (ruzi grass, rice straw or urea treated rice straw). Microbial biomass, net $^{15}N$ incorporation into cells, volatile fatty acid production, gas volume and rate of gas production increased linearly with increasing levels of cassava inclusion. There was also an effect of roughage source, with rice straw being associated with the lowest values for most parameters whilst similar values were obtained for ruzi grass and urea treated rice straw. The results suggest that microbial growth and fermentation rate increase as a function of readily available carbohydrate in the substrate mixture. A strong linear relationship between $^{15}N$ enrichment, total volatile fatty acid production and gas production kinetics support the suggestion of the use of the in vitro gas production system as a tool for screening feedstuffs as an initial stage of feed evaluation.

• 한국환경보건학회지
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• 제24권2호
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• pp.126-133
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• 1998

Effects of B(a)P on preimplantation mouse embryos in vitro were studied. Preimplantation mouse embryos were exposed to a concentration of 0.3, 1, 3 and 10 $\mu$M B(a)P for 72 hrs. The toxicological effects of B(a)P were evaluated by morphological observation of embryos up to the blastocyst stage, and by measuring DNA, RNA and protein synthesis by radioactive precursor incorporation. At 1 $\mu$M B(a)P did not affect preimplantation development but interfered with hatching and ICM formation. Suppressing effect of ICM formation was dose dependent. At the eight cell stage, the developmental rate was decreased at above 3 $\mu$M of B(a)P. At the blastocyst stage, attachment and trophoblast outgrowth were diminished at the 10 $\mu$M of B(a)P and ICM formation was decreased at 1 $\mu$M of B(a)P. Inner cell number of blastocyst was decreased dose dependently. So, number of ICM was one of the most sensitive and toxicological end point. The RNA incorporation rate of 0.1 $\mu ^3$H-uridine was dosedependent and the protein incroporation of 0.5 $\mu Ci ^{35}$S-methionine showed a significant decrease after 48 hrs. But the DNA incorporation rate of methyl-$^3$H thymidine was not affected. Our results suggested that B(a)P did not affect the DNA replication but transcription was inhibited by dose dependent manner. There delay of development during the blastocyst stage was mainly due to the inhibition of RNA synthesis followed by protein synthesis.

• Toxicological Research
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• 제4권1호
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• pp.37-45
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• 1988

Primary cultures of adult rat hepatocytes were used to study in vitro cytotoxic effects of T-2 toxin on liver cells. When T-2 toxin was added to the culture, a significant depression of the hormonal induction of ${\alpha}$-aminoisobutyric acid (AIB) uptake and tyrosine aminotransferase (TAT) activity was observed. However, T-2 toxin did not affect the uptake of ouabain into hepatocytes. Protein synthesis was inhibited by T-2 toxin, but RNA synthesis was not severely affected. The inhibitory effects of T-2 toxin on protein synthesis was diminished rapidly with culture time and the hepatocytes culture maintained control level of protein synthesis within 24 hrs.

• Asian-Australasian Journal of Animal Sciences
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• 제8권5호
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• pp.455-462
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• 1995

Arbor Acres broiler chickens (N=288) with an average initial weight of 59.4 g were fed diets varying in protein and lysine (80, 100, 120% of NRC; 100, 120% of NRC, 1984) in order to investigate the effects of supplemental chromium picolinate on growth performance, nutrient utilizability, carcass composition, serum traits and in vitro protein synthesis. Six replicates of eight chicks were grouped into one treatment Six chicks were sacrificed from each treatment for carcass analysis, and six additional chicks were chosen and dissected for in vitro culture of liver tissue. Body weight gain, feed intake, feed conversion, mortality, carcass composition and serum glucose, HDL/cholesterol ratio, serum triglyceride and serum nonesterified fatty acid appeared to be affected by either the level of dietary crude protein or lysine when supplemented with 200 ppb chromium picolinate (p < 0.05). Retained and secreted proteins in liver acinar cell cultured in vitro were not affected by dietary lysine level but affected by dietary protein level when added with 200 ppb chromium picolinate.

• Molecules and Cells
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• 제19권2호
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• pp.239-245
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• 2005

Factor for inversion stimulation (FIS), the Escherichia coli protein, is a positive regulator of the transcription of genes that encode stable RNA species, such as rRNA and tRNA. Transcription of the rnpB gene encoding M1 RNA, the catalytic subunit of E. coli RNase P, rapidly declines under stringent conditions, as does that of other stable RNAs. There are multiple putative FIS binding sites upstream of the rnpB promoter. We tested whether FIS binds to these sites, and if so, how it affects rnpB transcription. In vitro binding assays revealed specific binding of FIS to multiple sites in the rnpB promoter region. Interestingly, FIS bound not only to the upstream region of the promoter, but also to the region from +4 to +18. FIS activated rnpB transcription in vitro, but the level of activation was much lower than that of the rrnB promoter for rRNA. We also examined the effects of FIS on rnpB transcription in vivo using isogenic $fis^+$ and $fis^-$ strains. rnpB transcription was higher in the $fis^-$ than the $fis^+$ cells during the transitions from lag to exponential phase, and from exponential to stationary phase.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권5호
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• pp.311-315
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• 2002

By the in vitro combinatorial mutagenesis, which is a sequential reaction of PCR mutagenesis and in vitro coupled transcription/translation with Escherichia coli S30 extract, S65 and E222 of green fluorescent protein of Aequarea victoria were comprehensively changed to all possible combinations of amino acids, thus totally 400 mutant (including a wild type) proteins were simultaneously produced and their fluorescent properties were analyzed. Although a few mutations had been reported so far at the 222nd position, replacement E222 to all other19 amino acids gave fluorescent signal to the mutants by changing Ser 65 to Ala together. Among the mutants, replacement to G, A, S, Q, H and C gave relatively high fluorescence. The in vitro combinatorial mutagenesis, therefore, has been proved valuable for comprehensive structure-function studies of proteins.