• Journal of the Korean Home Economics Association
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• v.30 no.3
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• pp.91-99
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• 1992

Cell structure is based on proteins. Since the antibody is proteous substance, the continous low protein feeding decreases the resistance of host against pathogenic agents. The present study was designed to investigate the infectivity of protozoa to rats which were fed with variously prescribed diets. Experimental group was divided into 4 groups according to the level of casein in the diet, group I: casein 0%, group II: casein 5%, group III: casein 15%, group IV: casein 30%. Each animal was fed for 5 weeks followed by inoculation of protozoa in cecum and sacrified each 1 week later of the infection. Each diet group, non infected with protozoa was recognized as the control. Result are summerized as follows : 1. All the rats of group I died in 2∼4 weeks and 2 of 12 rats in group II were also died in the period. 2. The growth rate and FER were high in group III and IV compared with group II. Therefore low protein feeding decrease growth and feed efficaly ratio(FER). 3. The pH of caecal contents between the infected group and control showed no difference, but the values of group III and IV were higher than the group II. Low pH of the caecal contents provides a suitable condition for determining their susceptibility to Entameoeba histolytical trophozoite. 4. Amounts of serum total protein in group II, III and IV showed no significant difference with the control and infected group, but amounts in group III and IV were higher than the group II. Therefore, continuous low protein feeding decrease serum total protein. 5. Albumin, ${\alpha}$1, gloulin, ${\alpha}$2 globulin, ${\beta}$ globulin, ${\gamma}$ gloulin of group III and IV were all high to compare than the group II. Albumins of group III and IV of control was higher than infected group, but there was no difference in ${\gamma}$ globulin between the infected and control group.

• Korean Journal of Animal Reproduction
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• v.18 no.2
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• pp.133-139
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• 1994

Two human lactoferrin expression vectors(pCChcLf and pCChcLf-1) were constructed using rat $\beta$-casein gene and human lactoferrin cDNA. The recombinant DNAs containing human lactoferrin cDNA were microinjected into the fertilized eggs of hybrid mice (BDF1 : C57BL$\times$DBA) and the DNA-injected eggs were treansferred into the oviducts of foster mothers. Genomic DNAs were isolated from the tails of mice born from the microinjected eggs and analyzed by Southern blot analysis. As a result, 5 and 9 transgenic mice with CChcLf and CChcLf-1 gene were produced, respectively. To determine tissue-specificity of transgene expression, Northern blot analysis was performed. Female transgenic mice were killed at day 10 of lactation and total RNAs from various tissues were isolated. Based on Northern blot analysis, it was shown that transgene was mainly expressed in the mammary glands of transgenic mice. In addition, the human lactoferrin in milk was detected by enzyme-linked immunosorbent assay. For this study, milk was obtained from the mammary glands of the transgenic mice at day 10 of lactation. In line #2 of CChcLf and line #7 of CChcLf-1 transgenic mice, human lactoferrin was secreted into the milk at concentration levels of 340ng/ml and 60ng/ml, respectively.

• Reproductive and Developmental Biology
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• v.40 no.1
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• pp.7-13
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• 2016

Effectiveness of transgene transfer into genome is crucially concerned in mass production of the bio-pharmaceuticals using genetically modified transgenic animals as a bioreactor. Recently, the mammary gland has been considered as a potential bioreactor for the mass production of the bio-pharmaceuticals, which appears to be capable of appropriate post-translational modifications of recombinant proteins. The mammary gland tissue specific vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals. In this study, to minimize physiological disturbance caused by constitutive over-expression of the exogenous gene, we constructed new retrovirus vector system designed for mammary gland-specific expression of the hEPO gene. Using piggyBac vector system, we designed to express hEPO gene under the control of mammary gland tissue specific and lactogenic hormonal inducible goat ${\beta}$-casein or mouse Whey Acidic Protein (mWAP) promoter. Inducible expression of the hEPO gene was confirmed using RT-PCR and ELISA in the mouse mammary gland cells treated with lactogenic hormone. We expect the vector system may optimize production efficiency of transgenic animal and reduce the risk of global expression of transgene.

• Development and Reproduction
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• v.17 no.1
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• pp.1-8
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• 2013

Osteoprotegerin (OPG) is a secreted glycoprotein that regulates bone resorption by inhibiting differentiation and activation of osteoclast, thereby potentially useful for the treatment of many bone diseases associated with increased bone loss. In this study, we designed a novel cDNA expression cassette by modifying the potent and mammary gland-specific goat ${\beta}$-casein/hGH hybrid gene construct and examined human OPG (hOPG) cDNA expression in transgenic mice. Six transgenic mice all successfully expressed hOPG in their milk at the level of 0.06-2,000 ${\mu}g/ml$. An estimated molecular weight of the milk hOPG was 55 kDa in SDS-PAGE, which is the same as a naturally glycosylated monomer. This hOPG expression was highly specific to the mammary glands of transgenic mice. hOPG mRNA was not detected in any organs analyzed except mammary gland. Functional integrity of milk hOPG was evaluated by TRAP (tartrate-resistant acid phosphatase) activity assay in bone marrow cell cultures. OPG ligand (OPG-L) treatment increased TRAP activity by two fold but it was completely abolished by co-treatment with transgenic milk containing hOPG. Taken together, our novel cDNA expression cassette could direct an efficient expression of biologically active hOPG, a potential candidate pharmaceutical for bone diseases, only in the mammary gland of transgenic mice.

• Molecules and Cells
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• v.23 no.3
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• pp.340-348
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• 2007

Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi-phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.

• Journal of the Korea Organic Resources Recycling Association
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• v.2 no.2
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• pp.31-37
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• 1994

For a microbiological study of composting process, screening and assay method for biopolymer degrading enzymes and microorganisms were developed and for the study of the possibility of composting in anaerobic state, distribution of sulfate reducing bacteria which plays a final role in anaerobic degradation was investigated. Substrates used for the development of assay methods for biopolymer degradation are ${\beta}-glucan$, xylan, dextran, CMC(carboxy methly cellulose), casein, and collagen. These substrates were made insoluble by a cross-linking agent and linked with dye to make chromogenic substrates. ${\beta}-glucan$ and xylan substrates could substitute congo-red method for screening of polymer degrading microorganisms without damaging the colonies. Sulfate reducing bacteria contained in the sample sludge showed preference to lactic acid, propionic acid, butyric acid and formic acid and could use acetic acid and valeric acid.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.6
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• pp.1012-1022
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• 2020

Objective: Caseins and fatty acids of milk are synthesized and secreted by the epithelial cells of the mammary gland. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, has been shown to promote mammary development. This study was conducted to determine the effect of ATRA on casein synthesis and fatty acid composition in MAC-T cells. Methods: MAC-T cells were allowed to differentiate for 4 d, treated with ATRA (0, 1.0, 1.5, and 2.0 μM), and incubated for 3 d. We analyzed the fatty acid composition, the mRNA expression of casein and fatty acid synthesis-related genes, and the phosphorylation of casein synthesis-related proteins of MAC-T cells by gas chromatography, quantitative polymerase chain reaction, and western blotting, respectively. Results: In MAC-T cells, ATRA increased the mRNA levels of α_S1-casein and β-casein, janus kinase 2 (JAK2) and E74-like factor 5 of the signal transducer and activator of transcription 5 β (STAT5-β) pathway, ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 of the mammalian target of rapamycin (mTOR) pathway, inhibited the mRNA expression of phosphoinositide 3-kinase and eukaryotic initiation factor 4E of the mTOR pathway, and promoted the phosphorylation of STAT5-β and S6K1 proteins. Additionally, ATRA increased the de novo synthesis of fatty acids, reduced the content of long-chain fatty acids, the ratio of monounsaturated fatty acids to saturated fatty acids (SFA), the ratio of polyunsaturated fatty acids (PUFA) to SFA, and the ratio of ω-6 to ω-3 PUFA. The mRNA levels of acetyl-CoA carboxylase 1, fatty acid synthase, lipoprotein lipase, stearoyl-CoA desaturase, peroxisome proliferator-activated receptor gamma, and sterol regulatory element-binding protein 1 (SREBP1) were enhanced by ATRA. Conclusion: ATRA promotes the synthesis of casein by regulating JAK2/STAT5 pathway and downstream mTOR signaling pathway, and it improves the fatty acid composition of MAC-T cells by regulating SREBP1-related genes.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.87-87
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• 2002

Previously, we observed high level expression of goat β-casein/genomic hGH fusion gene in mammary gland of transgenic mice. To develop an expression vector to make a human granulocyte-colony stimulating factor (hG-CSF) protein efficiently produced in milk of transgenic animals, we designed a new bicistronic vector using the goat β-casein/genomic hGH fusion gene as regulation sequences for expression and internal ribosome entry site (IRES) as a mediator for second gene expression. This vector was constructed by insertion of encephalomyocarditis virus (EMCV) IRES-dependent second gene region coupled with hG-CSF cDNA into 3' untranslated region of an intact hGH gene. By microinjcetion, four transgenic mice were generated and three of them transmitted the bicistronic vector to their progeny. (omitted)

• Food Science of Animal Resources
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• v.39 no.5
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• pp.725-741
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• 2019

In the current study, we first investigated a method for directly transforming lactose into galacto-oligosaccharides (GOS) for manufacturing low-lactose and GOS-enriched skim milk (GSM) and then evaluated its prebiotic potential by inoculating five strains of Bifidobacterium spp. In addition, fermented GSM (FGSM) was prepared using a potentially probiotic Lactobacillus strain and its fermentation characteristics and antioxidant capacities were determined. We found that GOS in GSM were metabolized by all five Bifidobacterium strains after incubation and promoted their growth. The levels of antioxidant activities including radical scavenging activities and 3-hydroxy-3-methylglutaryl-CoA reductase inhibition rate in GSM were significantly increased by fermentation with the probiotic Lactobacillus strain. Moreover, thirty-nine featured peptides in FGSM was detected. In particular, six peptides derived from ${\beta}$-casein, two peptides originated from ${\alpha}s_1$-casein and ${\kappa}$-casein were newly identified, respectively. Our findings indicate that GSM can potentially be used as a prebiotic substrate and FGSM can potentially prevent oxidative stress during the production of synbiotic fermented milk in the food industry.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.371-378
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• 1997

Human lactoferrin (hLF) was expressed in the mammary gland of transgenic mice. Expresion of hLF was achieved by palcing its cDNA under the control of bovine $\beta$-casein gene. To improve the hLF expression level, two artificial introns were introduced into the expression vector. One intron is a hybrid-splice consisting of bovine $\beta$ casein intron 1 and rabbit $\beta$-casem intron II. The other intron is a DNA fragment spanning intron 8 of bovine $\beta$ casein gene. Trans sgenic mice were developed which expressed hLF in their milk. Twenty lines of transgenic mice were produced. hLF was present in the milk at concentrations of 1 ~ 200 ${\mu}\textrm{g}$ / ml. hLF RNA was only detected in the mammary gland of transgenic mice. The expressed RNA was cor r rectly spliced at the exon /intron junctions. To generate transgenic cows secreting active hLF in their milk, we transferred the DNA-injected bovine embryos to recipient heifers by surgical a and non-surgical methods out of 68 embryos transferred to 51 recipients by surgical or non-surgical method, 7 calves were normally born. Effect of embryo quality of DNA-injected blastocysts on pregnancy rate after transfer was investig a ated. Higher pregnancy rate of (38.9%) DNA-injected embryos was shown in excellent embryos. Pregnancy rates in the groups of good a and fair embryos were 15.4 and 14.3%, respectively. Effect of culture period of DNA-injected b bovine embryos on pregnancy rate after transfer was investigated. When Day-6 blastocysts of cuI ture were transferred, there was no pregnancy. Pregnancy rates of Day-7 and -8 blastocysts were 28.6 and 33.3%, respectively. There was no difference on pregnancy rate between Day-7 a and -8 bovine blastocysts after DNA injection. Thus, we established the techniques for transfer a and culture of DNA-injected bovine embryos. In a addition, factors affecting the pregnancy rate of DNA-injected embryos after transfer were investigated .