• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.878-885
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• 2017

Objective: Glucose is an essential fuel in the energy metabolism and synthesis pathways of all mammalian cells. In lactating animals, glucose is the major precursor for lactose and is a substrate for the synthesis of milk proteins and fat in mammary secretory (alveolar) epithelial cells. However, clear utilization of glucose in mammary cells during lactogenesis is still unknown, due to the lack of in vitro analyzing models. Therefore, the objective of this study was to test the reliability of the mammary alveolar (MAC-T) cell as an in vitro study model for glucose metabolism and lactating system. Methods: Undifferentiated MAC-T cells were cultured in three types of Dulbecco's modified Eagle's medium with varying levels of glucose (no-glucose: 0 g/L, low-glucose: 1 g/L, and high-glucose: 4.5 g/L) for 8 d, after which differentiation to casein secretion was induced. Cell proliferation and expression levels of apoptotic genes, Insulin like growth factor-1 (IGF1) receptor, oxytocin receptor, ${\alpha}S1$, ${\alpha}S2$, and ${\beta}$ casein genes were analyzed at 1, 2, 4, and 8 d after differentiation. Results: The proliferation of MAC-T cells with high-glucose treatment was seen to be significantly higher. Expression of apoptotic genes was not affected in any group. However, expression levels of the mammary development related gene (IGF1 receptor) and lactation related gene (oxytocin receptor) were significantly higher in the low-glucose group. Expressions of ${\alpha}S1-casein$, ${\alpha}S2-casein$, and ${\beta}-casein$ were also higher in the low-glucose treated group as compared to that in the no-glucose and high-glucose groups. Conclusion: The results demonstrated that although a high-glucose environment increases cell proliferation in MAC-T cells, a low-glucose treatment to MAC-T cells induces higher expression of casein genes. Our results suggest that the MAC-T cells may be used as an in vitro model to analyze mammary cell development and lactation connected with precise biological effects.

• Journal of Nutrition and Health
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• v.18 no.4
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• pp.318-327
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• 1985

This study was designed to investigate the effect of early protein undernutrition of rats on later susceptibility to lead poisoning. Weanling rats, weighing about 55 grams, were malnourished by feeding low protein diets ( 12% and 5% casein diet ) during the four weeks of protein deprivation period. For the following 5 weeks, the malnourished rats were fed with 25% casein diet as rehabilitation diet. After the rehabilitation period, all rats were fed with 25% casein diet and the drinking water containing 2000ppm-Pb during the four weeks. The results obtained were as follows ; 1 ) Feed intake, body weight gain and food efficiency ratio were reduced in all rats exposed to lead, especially in rats fed with 5% casein diet during the four weeks of weanling period. 2 ) Enlargement of kidney and spleen were observed in all rats exposed to lead and were more remarkable in rats fed with 5% casein diet. 3 ) In rats exposed to lead, activity of serum glutamic oxaloacetic transaminase ( S- GOT ) was significantly increased by increasing the degree of early protein deprivation, whereas hematocrit value was significantly decreased. Activity of serum glutamic pyruvic transaminase ( S- GPT ) was shown a tendency to increase by increasing the degree of early protein deprivation, but the difference was not significant. 4) In rats exposed to lead, the amount of lead accumulation in kidney was increased by increasing the degree of early protein deprivation. The significant increase was shown in rats fed with 5% casein diet during the four weeks of weanling period. The results of the experiment suggest that early protein undernutrition, even after some period of rehabilitation, may enhance the later susceptibility to lead poisoning.

• Korean Journal of Food Science and Technology
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• v.30 no.1
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• pp.218-223
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• 1998

Casein was hydrolyzed by alcalase to produce iron binding peptide (IBP). IBP was effectively separated from casein hydrolysates by immobilized $Fe^{3+}$ affinity chromatography and further purified by reverse phase chromatography. $25,\;50\;and\;100\;{\mu}g/mL$ of IBP solubilized $4.2,\;5.7\;and\;7.1\;{\mu}g$ of ferric at duodenum condition $(pH\;6,\;37^{\circ}C)$, respectively. According to the result of MALDI analysis, molecular weight of IBP was determined to 2,175 dalton. IBP was mainly composed of proline (24.5 mol%), lysine (15.7 mol%), and glutamine or glutamic acid (14.9 mol%) and its N-terminal sequence was Met-Ala-Pro-Lys-His. According to the information obtained from molecular weight, amino acids composition and N-terminal sequence of IBP, it was evident that IBP was from f102-119 of ${\beta}-casein$.

• Asian-Australasian Journal of Animal Sciences
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• v.5 no.1
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• pp.123-128
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• 1992

In Experiment 1, when fasted chicks were fed diets containing various sources of protein for 3 days, the activities of lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthetase, citrate cleavage enzyme and malic enzyme) in the liver of growing chicks were significantly lower in the soybean protein or gluten diet than in the casein or fish protein diet. Triglycride contents of the liver and plasma of chicks fed the casein or fish protein diet were significantly lower than that of those fed soybean protein or gluten diet. In Experiment 2, the effects of dietary amino acid mixture simulating casein or protein on the activities of hepatic lipogenic enzymes were examined. The activities of acetyl-CoA carboxylase and fatty acid synthetase in the liver of chicks fed the casein diet were significantly higher than that of those fed the soybean protein diet or two diets of amino acid mixtures. Furthermore, there were no significant differences between the two diets of amino acid mixture based on casein or soybean protein. However, the activities of malic enzyme and citrate cleavage enzyme tended to be lower in the soybean-type amino acid diet than in the casein-type amino acid diet. Thus, some effects can be ascribed to the protein itself and some to the amino acid composition of the protein sources.

• 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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.31 no.2
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• pp.263-270
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• 2002

In the recent studies, many researchers are interested in foods as functional components rather than nutrient sources. Cow's milk is considered as an excellent food sources because of its many nutrients. Casein is a major milk protein and has been reported to have hyperlipidemic and hypercholesterolemic effects. But several reporters have suggested that peptide fractions and hydrolysate of casein have hypolipidemic effects differing from intact protein, casein. Therefore, the objective of the study was to investigate how the casein peptide fractions affect lipid metabolism in rats fed normal or high fat diets. The peptide fractions and hydrolysate of casein were obtained by casein hydrolysis with trypsin. The male rats (Sprague-Dawley), weighing approximately 150 g, were fed each experimental diet containing casein (CAS), casein hydrolysate (CH), casein hydrolysate precipitate (Cpt) and two kinds of peptide fractions (CL & CB) for three weeks, respectively. In the exit I, the male rats were fed normal fat diets (7% soybean oil & cholesterol-free; Expt. I), and in the expt II, fed high fat diets (18% beef tallow & 1% cholesterol; Expt. II). Crude protein contents were calculated from nitrogen contents. Amino acid composition of each fraction was also analyzed. The concentration of total lipid, total cholesterol and triglyceride in serum, liver and feces were measured. As the results of study, tole rats fed peptide fractions with normal fat diets (Expt. I) had no effects on total lipid, total cholesterol and triglyceride concentration in serum and liver and fecal excretion. However, in the rats fed hydrophobic casein peptide fractions (CB) with high fat diet, fecal lipids excretion were significantly increased and the lipids concentration of serum and those of liver tended to decrease, numerically.

• KSBB Journal
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• v.5 no.3
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• pp.241-246
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• 1990

ATP analogs were immobilized or bovine caseins by the action of transglutaminase. The ATP analogs immobilized on the caseins were enzymatically active and interconverten by kinases. The immobilized ATP was dephosphorylated to the corresponding ADP by hexokinase and rephosphorylated to the ATP in solid form by acetate kinase. Under the conditions chosen, about 55% of the immobilized ATP was dephosphorylated and about 80% of the resulted ADP was rephosphorylated. Bovine $\beta$-casein was more useful than $\alpha$sf-casein as a carrier and C8-substituted ATP analognwas more effective than N6-substituted one in immobilization. Michaelis constant of C8-substituted ATP analog immobilized on $\beta$-casein was similar to that of free form of ATP and that of ATP analog. The immobilized ATP was much more stable than free ATP and its analog, while maximum velocity was reduced to 37% of the free ATP analog. The immobilized ATP was recovered almost completely by calcium precipitation.

• Korean Journal of Agricultural Science
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• v.36 no.1
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• pp.111-122
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• 2009

When the cattail pollen was identified by using fibrinolytic agents, we found that the fibrinolytic activity was controlled by an enzyme. Therefore, for determining the fibrinolytic activity of cattail pollen, the fibrinolytic enzyme in cattail pollen was purified by gel filtration using DEAE-cellulose, Sephadex G-150 and HPLC. Also, its purity was certified by polyacrylamide gel electrophoresis, and its physico-chemical properties, such as pH and temperature stabilities and effects of metal, inhibitors and substrates, were examined. The specific activity, purification fold, and molecular weight of the enzyme were 38U/mg, 86.4,and 75kDa, respectively. The optimum pH for the purified enzyme was at 4.0 and it was stable at pH 4.0-6.0. The optimum temperature was $55^{\circ}C$ and it was stable at $30-60^{\circ}C$. But the enzyme began to be inactivated at $70^{\circ}C$ and its activity was totally lost at temperatures above $80^{\circ}C$. As for substrate specificity, the enzyme was most effective in dissolving fibrin, followed by whole casein, ${\kappa}$-casein, ${\alpha}$-casein, ${\beta}$-casein, and BSA. With casein as the substrate, Km value was found to be 0.44mM and the enzyme showed a high affinity for casein. As for the metal ions affecting enzyme activity, $K^+$, $Na^+$, and $Mg^{2+}$ had no effect on enzyme reaction while $Zn^{2+}$ and $Fe^{2+}$ showed potent inhibitory activity. Judging from the fact that the purified enzyme was also strongly inhibited by PMSF, iodoacetic acid, and SDA, it assumed to be a serine protease.

• Journal of Animal Science and Technology
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• v.44 no.6
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• pp.801-808
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• 2002

Proteolytic activities of some commercial milk clotting enzymes(rennet, trypsin, pepsin, papain W-40, neutrase 1.5 and protease S) in bovine skim milk containing 0.02% $CaCl_2$ were determined by measuring DH(Degree of Hydrolysis), NPN(Non Protein Nitrogen) and by comparing patterns of SDS-PAGE(Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis). The DH of microbial enzymes(neutrase 1.5 and protease S) and trypsin in bovine skim milk were higher than those of pepsin and papain W-40. The amounts of NPN in the milk treated with trypsin and the other animal enzymes(rennet and pepsin) showed the highest and lowest degrees of proteolysis, respectively. SDS-PAGE showed that trypsin and protease S hydrolyzed $\alpha$-lactalbumin and papain W-40 hydrolyzed $\beta$-lactoglobulin slightly, while neutrase 1.5 hydrolyzed both $\alpha$-lactalbumin and $\beta$-lactoglobulin after treating for 90 min. Trypsin and protease S easily hydrolyzed ${\alpha}_s$-casein and $\beta$-casein, which were not hydrolyzed by rennet. Papain W-40 hydrolyzed $\kappa$-casein more than rennet as shown in SDS-PAGE. Based on the results of the experiments, the DH and NPN of trypsin, neutrase 1.5 and protease S were shown to be higher than those of the other enzymes. The SDS-PAGE patterns of papain W-40 and neutrase 1.5 were similar with that of rennet.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.3
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• pp.326-331
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• 2005

A rumen simulated continuous culture (RSCC) system was used to study the influence of supplementation of the three different types of protein sources such as urea, casein and soy protein on rumen microbial synthesis in terms of rumen microbial synchronization. The urea treatment showed the highest pH value. Ammonia nitrogen concentration was rapidly increased after feeding and not significantly different in the urea treatment (13.53 mg/100 ml). Protozoa numbers were not significantly different for soy protein and casein treatment compared to urea treatments during incubation. The average concentration of total VFA (mMol) was not detected with significant difference among treatments, but iso-butyrate production showed the highest for soy protein treatment among treatments (p<0.001). The lowest concentration in total iso-acids (iso-butyrate and iso-valerate) production was observed in urea treatment. The soy protein treatment showed no significantly change in acetate/propionate. The amounts of dry matter (DM) out flow showed no significant difference among treatments. Organic matter (OM) flow was the highest for urea treatments and the lowest for casein treatment (p<0.03). The nitrogen flow for casein treatment was not significantly different from other treatments. The efficiency of microbial protein synthesis in terms of microbial nitrogen (MN) synthesis (g MN/kg ADOM) digested in the rumen was highest for casein treatment (58.53 g MN/kg ADOM) compared to soy protein and urea (p<0.05). This result suggests that rumen ammonia releasing rate may influence on microbial protein synthesis in the rumen.