• BMB Reports
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• v.36 no.4
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• pp.390-398
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• 2003

The CDP/Cux transcription factor was previously shown to be proteolytically processed at the G1/S transition. In view of characterizing and eventually identifying the protease responsible for CDP/Cux processing, we have established an in vitro proteolytic processing assay. CDP/Cux recombinant proteins expressed in mammalian or bacterial cells were efficiently processed in vitro using as a source of protease either whole cell extracts, the nuclear or the cytoplasmic fraction. Processing was found to take place optimally at a lower pH, to be insensitive to variations in salt concentration, and to be inhibited by the protease inhibitors MG132 and E64D. Interestingly, the bacterially-produced substrate was more efficiently processed than the substrate purified from mammalian cells. Moreover, processing in vitro was more efficient when CDP/Cux substrates were purified from populations of cells enriched in the S phase than in the G1 phase of the cell cycle. Altogether, these results suggest that post-translational modifications of CDP/Cux in mammalian cells inhibits processing and contributes to the cell cycle-dependent regulation of processing. The in vitro processing assay described in this study will provide a useful tool for the purification and identification of the protease responsible for the processing of CDP/Cux.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.6
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• pp.783-791
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• 2019

Objective: This study was to evaluate the fermentation dynamics, structural and nonstructural carbohydrate composition and in vitro gas production of rice straw ensiled with lactic acid bacteria and molasses. Methods: Fresh rice straw was ensiled in 1-L laboratory silos with no additive control (C), Lactobacillus plantarum (L), molasses (M) and molasses+Lactobacillus plantarum (ML) for 6, 15, 30, and 60 days. After storage, the silages were subjected to microbial and chemical analyses as well as the further in vitro fermentation trial. Results: All additives increased lactic acid concentration, and reduced pH, dry matter (DM) loss and structural carbohydrate content relative to the control (p<0.05). The highest organic acid and residual sugar contents and lignocellulose reduction were observed in ML silage. L silage had the highest V-score with 88.10 followed by ML silage. L and ML silage improved in vitro DM digestibility as compared with other treatments, while in vitro neutral detergent fibre degradability (IVNDFD) was increased in M and ML silage (p<0.05). M silage significantly (p<0.05) increased propionic acid (PA) content and decreased butyric acid content and acetic acid/PA as well as 72-h cumulative gas production. Conclusion: The application of ML was effective for improving both the fermentation quality and in vitro digestibility of rice straw silage. Inclusion with molasses to rice straw could reduce in vitro ruminal gas production.

• Korean Journal of Organic Agriculture
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• v.26 no.2
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• pp.281-296
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• 2018

This study was conducted to evaluate the effect of different processing of rice on rumen fermentation in in vitro and in situ experiments. Different processing treatments (extruding, roasting, and steaming) were used in this study and all treatments were ground through a cyclone mill (Foss, Hillerød, Denmark) fitted with a 1 mm screen. Non-treated rice was considered to a control substrate. Then, all treatments were used in in vitro and in situ experiments. Total gas production and dry matter digestibility in control were lower than any other treatment at all incubation times (P<0.01). The lowest ammonia nitrogen ($NH_3-N$) concentration was observed in control among treatments at 6, 12, and 24 h incubation (P<0.01). Extruding had a highest total volatile fatty acids (VFA) concentration at 6, 12 h incubation (P<0.01) and Steaming exhibited a highest total VFA at 24 h (P<0.01). The lowest total VFA concentration was observed in control at 6, 12, and 24 h (P<0.01). In an in situ, The highest value of soluble fraction, degradation rates, effective degradability was observed in extruding (P<0.01). It was considered that feed processing increased dry matter digestibility, total VFA concentration, and decreased pH as well as $NH_3-N$ concentration indicating that processing may increase nutrient degradation of rice in the rumen.

• Journal of Microbiology and Biotechnology
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• v.33 no.11
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• pp.1513-1520
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• 2023

Kex2 protease (Kex2p) is a membrane-bound serine protease responsible for the proteolytic maturation of various secretory proteins by cleaving after dibasic residues in the late Golgi network. In this study, we present an application of Kex2p as an alternative endoprotease for the in vitro processing of recombinant fusion proteins produced by the yeast Saccharomyces cerevisiae. The proteins were expressed with a fusion partner connected by a Kex2p cleavage sequence for enhanced expression and easy purification. To avoid in vivo processing of fusion proteins by Kex2p during secretion and to guarantee efficient removal of the fusion partners by in vitro Kex2p processing, P₁', P₂', P₄, and P₃ sites of Kex2p cleavage sites were elaborately manipulated. The general use of Kex2p in recombinant protein production was confirmed using several recombinant proteins.

• Journal of Life Science
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• v.11 no.2
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• pp.94-98
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• 2001

Type I signal peptidase cleaves the signal sequence from the amino terminus of membrane and secreted proteins afters these protein insert across the membrane. This enzyme serves as a potential target for the development of novel antibacterial agents due to its unique physiological and biochemical properties. Despite considerable research, the signal peptidase assay still remains improvement to provide further understanding of the mechanism and high-throughput inhibitor screening of this enzyme. In this paper, three known signal peptidase assays are tested with an E. coli D276A mutant signal peptidase to distinguish the sensitivity of each assays. In vitro assay using the procoat synthesized by in vitro transcription translation shows that the D276A signal peptidase I was inactive while in vivo processing of pro-OmpA expressed in the temperature-sensitive E. coli strain IT41 as well as in vitro assay using pro-OmpA nuclease A substrate show that D276A signal peptidase I has activity like wild-type signal peptidase. These results suggest that in vitro assay using the pro-OmpA nuclease A and in vivo pro-OmpA processing assay are more sensitive monitors than in vitro assay using the pro-coat. In conculsion, caution should be used when interpreting the in vitro results using the procoat.

• Animal Bioscience
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• v.34 no.2
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• pp.223-232
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• 2021

Objective: To investigate the improvement in utilization efficiency of total mixed ration (TMR) on Tibetan plateau, TMR were ensiled with different additives. Methods: A total of 150 experimental silos were prepared in a completely randomized design to evaluate the six treatments: i) control (without additive), ii) Lactobacillus buchneri (L. buchneri), iii) acetic acid, iv) propionic acid, v) 1,2-propanediol; and vi) 1-propanol. After 90 days of ensiling, silos were opened for fermentation quality and in vitro analysis, and then subjected to an aerobic stability test for 14 days. Results: Treating with L. buchneri, acetic acid, 1,2-propanediol and 1-propanol decreased propionic acid contents and yeast number, whereas increased (p<0.05) pH, acetic acid and ethanol contents in the fermented TMR. Despite increased dry matter (DM) loss in the TMRs treated with 1,2-propanediol and 1-pronanol, additives did not affect (p>0.05) all in vitro parameters including gas production at 24 h (GP24), GP rate constant, potential GP, in vitro DM digestibility and in vitro neutral detergent fibre digestibility. All additives improved the aerobic stability of ensiled TMR to different extents. Specially, aerobic stability of the ensiled TMR were substantially improved by L. buchneri, acetic acid, 1,2-propanediol, and 1-propanol, indicated by stable pH and lactic acid content during the aerobic stability test. Conclusion: L. buchneri, acetic acid, 1,2-propanediol, and 1-propanol had no adverse effect on in vitro digestibility, while ensiling TMR with the additives produced more acetic acid and ethanol, subsequently resulting in improvement of aerobic stability. There is a potential for some fermentation boosting additives to enhance aerobic stability of fermented TMR on Tibetan plateau.

• Animal Bioscience
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• v.34 no.6
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• pp.1038-1048
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• 2021

Objective: The study aimed to evaluate the effect of storage time and formic acid (FA) on fermentation characteristics, epiphytic microflora, carbohydrate components and in vitro digestibility of rice straw silage. Methods: Fresh rice straw was ensiled with four levels of FA (0%, 0.2%, 0.4%, and 0.6% of fresh weight) for 3, 6, 9, 15, 30, and 60 d. At each time point, the silos were opened and sampled for chemical and microbial analyses. Meanwhile, the fresh and 60-d ensiled rice straw were further subjected to in vitro analyses. Results: The results showed that 0.2% and 0.6% FA both produced well-preserved silages with low pH value and undetected butyric acid, whereas it was converse for 0.4% FA. The populations of enterobacteria, yeasts, moulds and aerobic bacteria were suppressed by 0.2% and 0.6% FA, resulting in lower dry matter loss, ammonia nitrogen and ethanol content (p<0.05). The increase of FA linearly (p<0.001) decreased neutral detergent fibre and hemicellulose, linearly (p<0.001) increased residual water soluble carbohydrate, glucose, fructose and xylose. The in vitro gas production of rice straw was decreased by ensilage but the initial gas production rate was increased, and further improved by FA application (p<0.05). No obvious difference of FA application on in vitro digestibility of dry matter, neutral detergent fibre, and acid detergent fibre was observed (p>0.05). Conclusion: The 0.2% FA application level promoted lactic acid fermentation while 0.6% FA restricted all microbial fermentation of rice straw silages. Rice straw ensiled with 0.2% FA or 0.6% FA improved its nutrient preservation without affecting digestion, with the 0.6% FA level best.

• Journal of Biomedical Engineering Research
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• v.19 no.4
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• pp.369-377
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• 1998

This paper describes the results of investigations comparing the relative in vitro responses of different signal processing algorithms for laser Doppler flowmetry(LDF) using self-mixing effect of laser diode(LD). A versatile laser Doppler system is described which enabled complex signal processing to be implemented relatively simply using digital analysis. The flexibility of the system allowed a variety of processing algorithms to be studied by simply characterising the algorithm of interest under software control using a personal computer. Two in-vitro physical models are also presented which was used to maintain reproducible fluid flows Flows of particles were studied in two physical models using a 780nm laser diode source. The results show that frequency weighted algorithms(first and second moments, rate to zero moment) are responsive to particle velocity more than concentration, whereas non-weighted algorithm (zero moment responds to concentration and velocity.

• Journal of Animal Science and Technology
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• v.62 no.5
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• pp.741-752
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• 2020

Herein, the in vitro protein digestibility of lyophilized Protaetia brevitarsis larvae flour with and without defatting using 70% ethanol was compared with beef loin. Proximate analysis showed that the defatted larvae contained the highest protein content (p < 0.05). The viable counts of total aerobic bacteria, Escherichia coli, and coliform bacteria decreased significantly after defatting the larval samples with 70% ethanol (p < 0.05). Measurement of α-amino group content and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed higher amounts of low molecular weight proteins in the larvae compared to beef loin (p < 0.05). After in vitro digestion, the degree of protein hydrolysis of the digesta was higher for both larvae samples compared to beef loin (p < 0.05). No change was observed in the in vitro larval protein digestibility after defatting. These results highlight the excellent protein digestibility of P. brevitarsis larvae with high protein content. Defatting insect flour with 70% ethanol could enhance microbial safety while maintaining excellent protein digestibility.

• Nutritional Sciences
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• v.3 no.2
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• pp.71-76
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• 2000

Effects of extrusion processing on zinc bioavailability in a product made from 85% semolina and 15% soy protein concentrate were determined using in vitro and in vivo techniques. Soluble and dialyzable zinc contents of the extruded product were 15.3 and 13.0 $\mu$g/g, respectively, compared to 15.6 to 15.0$\mu$g/g in the raw (unprocessed) ingredients. Zinc bioavailability in diets in which all of the zinc (14ppm) was provided either by the extruded product or by its raw ingredients was determined in two groups of male Sprague Dawley rats. No differences were found in concentrations of zinc in plasma, liver and femur between rats fed the two experimental diets. Apparent zinc digestibility was similar for both diets. These results are consistent with the in vitro results showing no effect of extrusion processing on zinc bioavailability.