• Food Science of Animal Resources
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• v.37 no.6
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• pp.940-947
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• 2017

Goat milk has a protein composition similar to that of breast milk and contains abundant nutrients, but its use in functional foods is rather limited in comparison to milk from other sources. The aim of this study was to prepare a goat A2 ${\beta}$-casein fraction with improved digestibility and hypoallergenic properties. We investigated the optimal conditions for the separation of A2 ${\beta}$-casein fraction from goat milk by pH adjustment to pH 4.4 and treating the casein suspension with calcium chloride (0.05 M for 1 h at $25^{\circ}C$). Selective reduction of ${\beta}$- lactoglobulin and ${\alpha}_s$-casein was confirmed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. The hypoallergenic property of A2 ${\beta}$-casein fraction was examined by measuring the release of histamine and tumor necrosis factor alpha from HMC-1 human mast cells exposed to different proteins, including A2 ${\beta}$-casein fraction. There was no significant difference in levels of both indicators between A2 ${\beta}$-casein treatment and the control (no protein treatment). The A2 ${\beta}$-casein fraction is abundant in essential amino acids, especially, branched-chain amino acids (leucine, valine, and isoleucine). The physicochemical properties of A2 ${\beta}$-casein fraction, including protein solubility and viscosity, are similar to those of bovine whole casein which is widely used as a protein source in various foods. Therefore, the goat A2 ${\beta}$-casein fraction may be useful as a food material with good digestibility and hypoallergenic properties for infants, the elderly, and people with metabolic disorders.

• KSBB Journal
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• v.17 no.2
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• pp.162-168
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• 2002

Human ferritin H- and L-chain genes(hfH and hfL) were cloned into the yeast shuttle vector YEp352 with various promoters, and the vectors constructed were used to transform Saccharomyces cerevisiae 2805. Three different promoters fused to hfH and hfL were used: galactokinase 1 (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase(GPD) promoter and alcohol dehydrogenase 1(ADH1 ) promoter. SDS-polyacrylamide gel electrophoresis and Western blotting analyses displayed expression of the introduced hfH and hfL. In the production of both ferritin H and L subunits GAL1 promoter was more effective than GPD promoter or ADH1 promoter. Ferritin H and L subunits produced in S. cerevisiae were spontaneously assembled into its holoproteins as proven on native polyacrylamide gels. Both recombinant H and L-chain ferritins were catalytically active in forming iron core. When the cells were cultured in the medium containing 10 mM ferric citrate, the cell-associated concentration of iron was 174.9 $\mu\textrm{g}$ Per gram(dry cell weight) for the recombinant yeast YG-L and 148.8 $\mu\textrm{g}$ Per gram(dry cell weight) for the recombinant yeast YG-L but was 49.4 $\mu\textrm{g}$ Per gram(dry cell weight) in the wild type, indicating that the iron contents of yeast is improved by heterologous expression of human ferritin H-chain or L-chain genes.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.534-541
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• 2009

The aim of this study was to evaluate the effects of gamma irradiation (${\gamma}$-irradiation) at doses of 15, 30 and 45 kGy on chemical composition, anti-nutritional factors, ruminal dry matter (DM) and crude protein (CP) degradibility, in vitro CP digestibility and to monitor the fate of true proteins of full-fat soybean (SB) in the rumen. Nylon bags of untreated or ${\gamma}$-irradiated SB were suspended in the rumens of three ruminally-fistulated bulls for up to 48 h and resulting data were fitted to a nonlinear degradation model to calculate degradation parameters of DM and CP. Proteins of untreated and treated SB bag residues were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Digestibility of rumen undegraded CP was estimated using the three-step in vitro procedure. The chemical composition of raw and irradiated soybeans was similar. Results showed that phytic acid in ${\gamma}$-irradiated SB at dose of 30 kGy was eliminated completely. The trypsin inhibitor activity of 15, 30 and 45 kGy ${\gamma}$-irradiated SB was decreased (p<0.01) by 18.4, 55.5 and 63.5%, respectively. From in sacco results, ${\gamma}$-irradiation decreased (p<0.05) the washout fractions of DM and CP at doses of 30 and 45 kGy, but increased (p<0.05) the potentially degradable fractions. Gamma irradiation at doses of 15, 30 and 45 kGy decreased (p<0.05) effective degradability of CP at a rumen outflow rate of 0.05 $h^{-1}$ by 4.4, 14.4 and 26.5%, respectively. On the contrary, digestibility of ruminally undegraded CP of irradiated SB at doses of 30 and 45 kGy was improved (p<0.05) by 12 and 28%, respectively. Electrophoretic analysis of untreated soybean proteins incubated in the rumen revealed that ${\beta}$-conglycinin subunits had disappeared at 2 h of incubation time, whereas the subunits of glycinin were more resistant to degradation until 16 h of incubation. From the SDS-PAGE patterns, acidic subunits of 15, 30 and 45 kGy ${\gamma}$-irradiated SB disappeared after 8, 8 and 16 h of incubation, respectively, while the basic subunits of glycinin were not degraded completely until 24, 48 and 48 h of incubation, respectively. It was concluded that ${\gamma}$-irradiated soybean proteins at doses higher than 15 kGy could be effectively protected from ruminal degradation.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.11
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• pp.1540-1545
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• 2012

Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism, but the profile of proteins associated with progesterone synthesis in cyclic and pregnant corpus luteum (CL) is not well-known in cattle. In Experiment 1, plasma progesterone level was monitored in cyclic cows (n = 5) and pregnant cows (n = 6; until d-90). A significant decline in the plasma progesterone level occurred at d-19 of cyclic cows. Progesterone level in abbatoir-derived luteal tissues was also determined at d 1 to 5, 6 to 13 and 14 to 20 of cyclic cows, and d-60 and -90 of pregnant cows (n = 5 each). Progesterone level in d-60 CL was not different from those in d 6 to 13 CL and d-90 CL, although the difference between d 6 to 13 and d-90 was significant. In Experiment 2, protein expression pattern in CL at d-90 (n = 4) was compared with that in CL of cyclic cows at d 6 to 13 (n = 5). Significant changes in the level of protein expression were detected in 32 protein spots by two-dimensional polyacrylamide gel electrophoresis (2-DE), and 23 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six proteins were found only in pregnant CL, while the other 17 proteins were found only in cyclic CL. Among the above 6 proteins, vimentin which is involved in the regulation of post-implantation development was included. Thus, the protein expression pattern in CL was disorientated from cyclic luteal phase to mid pregnancy, and alterations in specific CL protein expression may contribute to the maintenance of pregnancy in Korean native cows.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.5
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• pp.736-742
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• 2017

Objective: Experiments were conducted to clone the sequence of Wild Argali short palate, lung and nasal epithelium clone 1 (SPLUNC1) cDNA, and to lay the foundation for further study the biological function of Wild Argali SPLUNC1. Methods: The complete sequence of Wild Argali SPLUNC1 cDNA was generated by rapid amplification of cDNA ends. The entire coding sequence was inserted into the pPIC9K vector and expressed in Pichia pastoris (P. pastoris) GS115. The recombinant SPLUNC1 protein was detected by Western blot and purified by $Ni^{2+}$ chelate affinity chromatography. The test of effect of the protein on Mycoplasma ovipneumoniae (MO) was performed with real-time polymerase chain reaction. Results: The Wild Argali SPLUNC1 cDNA was 1,076 bp with an open reading frame of 768 bp, which encoded a 26.49 kDa protein composed of 255 amino acids. Its amino acid sequence shared 98.4%, 96.9%, 94.5%, 90.2%, 80.8%, 78.4%, 78.3%, 72.5%, 72.3%, 68.8% identity with those of SPLUNC1 cDNA from Ovis aries (accession no. NP_001288334.1), Capra hircus (accession no. XP_005688516.1), Pantholops hodgsonii (accession no. XP_005979709.1), Bos taurus (accession no. NP_776851.1), Felis catus (accession no. XP_006929910.1), Homo sapiens (accession no. NP_001230122.1), Sus scrofa (accession no. NP_001005727.1), Chinchilla lanigera (accession no. NP_001269294.1), Mus musculus (accession no. NP_035256.2), and Rattus norvegicus (accession no. NP_742028.1), respectively. The recombinant protein corresponded to the expected molecular mass of 25.47 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was detected in the supernatant of P. pastoris, and it could be purified. The results from the test of inhibition effect of argali recombinant SPLUNC1 protein on MO showed that the product could inhibit MO very well (p<0.01). Conclusion: The amino acid sequence of Wild Argali SPLUNC1 was different from other organisms. The recombinant SPLUNC1 protein has good biological activity.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.1
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• pp.125-131
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• 2015

Currently, taxol is mainly extracted from the bark of yews; however, this method can not meet its increasing demand on the market because yews grow very slowly and are a rare and endangered species belonging to first-level conservation plants. Recently, increasing efforts have been made to develop alternative means of taxol production; microbe fermentation would be a very promising method to increase the production scale of taxol. To determine the activities of the taxol extracted from endophytic fungus N. sylviforme HDFS4-26 in inhibiting the growth and causing the apoptosis of cancer cells, on comparison with the taxol extracted from the bark of yew, we used cellular morphology, cell counting kit (CCK-8) assay, staining (HO33258/PI and Giemsa), DNA agarose gel electrophoresis and flow cytometry (FCM) analyses to determine the apoptosis status of breast cancer MCF-7 cells, cervical cancer HeLa cells and ovarian cancer HO8910 cells. Our results showed that the fungal taxol inhibited the growth of MCF-7, HeLa and HO8910 cells in a dose-and time-dependent manner. IC50 values of fungal taxol for HeLa, MCF-7 and HO8910 cells were $0.1-1.0{\mu}g/ml$, $0.001-0.01{\mu}g/ml$ and $0.01-0.1{\mu}g/ml$, respectively. The fungal taxol induced these tumor cells to undergo apoptosis with typical apoptotic characteristics, including morphological changes for chromatin condensation, chromatin crescent formation, nucleus fragmentation, apoptotic body formation and G2/M cell cycle arrest. The fungal taxol at the $0.01-1.0{\mu}g/ml$ had significant effects of inducing apoptosis between 24-48 h, which was the same as that of taxol extracted from yews. This study offers important information and a new resource for the production of an important anticancer drug by endofungus fermentation.

• Interdisciplinary Bio Central
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• v.2 no.4
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• pp.12.1-12.7
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• 2010

The National Bio Resource Project-Rat (NBRP-Rat) comprises the largest bank of laboratory rat (Rattus norvegicus) strains in the world. Its main focus is to develop infrastructure that will facilitate the systematic collection, preservation, and provision of rat strains. To breed effectively more than 180 rat strains in living stock, we establish the genetic control system in which a systematic set of genetic diagnoses and genetic monitoring are included. Genetic monitoring is performed by using 20 polymorphic markers. Monitoring is carried out when a living animal stock is re-established by using cryopreserved embryos or sperm or when a rat strain is first introduced to the NBRP-Rat by a depositor. Additional monitoring is then carried out on each strain every two years. Genetic diagnosis is performed largely by employing the Amp-FTA method. Protocols which detail how to perform a genetic diagnosis of 11 transgenes and 24 mutations have been made. Among the mutations, nine can be detected by simple gel electrophoresis of the PCR products, 11 by restriction enzyme treatment of the PCR products, and four by direct PCR product sequencing. Using this genetic control system, the NBRP-Rat can guarantee the genetic quality of its rat strains.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.6
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• pp.775-778
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• 2006

Sarabi cows (n = 136) from the Sarabi Breeding Station were genotyped at bovine lymphocyte antigen (BoLA)-DRB3.2 locus by a genotyping system that used the polymerase chain reaction and restriction fragment length polymorphism. Genomic DNA was extracted from whole blood samples. A two-step polymerase chain reaction was carried out in order to amplify a 284 base-pair fragment of target gene. Nested-PCR products were digested with three restriction endonuclease enzymes RsaI, BstYI and HaeIII. Digested fragments were analyzed by polyacrylamide gel electrophoresis. Twenty-six BoLA-DRB3.2 alleles were identified with frequencies ranging from 0.4 to 15.1%. Six new allele types observed in this study have not been reported previously. Identified alleles include: BoLA-DRB3.$2^*1$, $^*2$, $^*4$, $^*6$, $^*8$, $^*12$, $^*13$, $^*14$, $^*15$, $^*16$, $^*17$, $^*23$, $^*24$, $^*25$, $^*28$, $^*32$, $^*34$, $^*35$, $^*36$, $^*37$, $^*42$, $^*46$, $^*51$, $^*kba$, $^*laa$ and $^*vaa$. Their frequencies were found to be 0.4, 0.4, 0.7, 11.4, 1.1, 1.8, 2.9, 2.2, 4.4, 9.6, 1.1, 13.6, 0.4, 0.4, 1.1, 0.7, 0.4, 6.2, 2.2, 3.7, 1.1, 7.7, 1.5, 15.1, 2.6 and 7.3% respectively. The six most frequent alleles (DRB3.2 $^*6$, $^*16$, $^*23$, $^*46$, $^*kba$ and $^*vaa$) accounted for 64.7% of the alleles in the population of this herd. Numerous studies on this locus, covering different breeds, has revealed the existence of various alleles in this locus, and new investigations have introduced novel alleles. With respect to the high number of the observed alleles in this survey and the novelty of some alleles with no previous record of reporting, it is plausible to conclude that the BoLA-DRB3.2 locus is highly polymorphic in Iranian native Sarabi cows.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.9
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• pp.1330-1338
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• 2008

This study was conducted to investigate the effects of Acanthopanax senticosus extract (ASE) as a dietary additive on gut microflora in weaned piglets. A total of sixty pigs were weaned at 21 d of age (BW = $5.64{\pm}0.23kg$) and allocated on the basis of BW and litter to three dietary treatments in a randomized complete block design. The dietary treatments were: control group (basal diet), antibiotics group (basal diet+0.02% colistin), and ASE group (basal diet+0.1% ASE). On d 7, 14 and 28 after consuming the experimental diets, five piglets per group were sacrificed and then the contents from the jejunum, ileum and cecum were collected to determine changes in the microbial community by using a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique and estimating the contents of Lactobacillus and E. coli by in vitro culturing methods. The results showed that the ASE promoted the microflora diversity in the cecum. Enumeration of bacteria in the gut contents showed that the number of Lactobacillus increased (p<0.05), while that of E. coli decreased (p<0.05) when compared with the other 2 groups as the days of age progressed post-weaning. These findings suggested that the ASE, as a substitute for dietary antimicrobial products, could improve the development of the normal gut microflora and suppress bacterial pathogens, and effectively promote a healthy intestinal environment.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.1
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• pp.126-133
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• 2016

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.