BACKGROUND/OBJECTIVES: Soy isoflavones are structurally similar to estrogen and bind to estrogen receptors, suggesting that they exhibit estrogenic activities; therefore, they are referred to as phytoestrogens. Fermentation may affect the bioavailability of isoflavones altering soy isoflavone glycosides in the form of aglycones. Thus, this study investigated the effects of fermented soybeans by Rhizopus oligosporus on bone metabolism in both young rats as a pilot test and in ovariectomized (ovx) old rats as a model of menopause. MATERIALS/METHODS: In the pilot test, a total of 24 seven-week-old female Sprague-Dawley (SD) rats were fed one of three diets for a period of four weeks: casein, unfermented soybean product, or fermented soybean product by R. oligosporus. In the ovx rat model, 20-week-old SD rats weighing 260-290 g underwent either sham-operation (n = 10) or bilateral ovariectomy (n = 30) and were then fed the AIN-93M diet for one week. Thereafter, rats were fed sham-casein, ovx-casein, ovx-soybean, or ovx-fermented soybean diet for five weeks. After decapitation, femoral bones were isolated and preserved in 9% formalin for assessment of bone mineral density (BMD), bone mineral content (BMC), and bone-breaking strength (BBS). RESULTS: Ovx rats showed significantly increased weight gain and decreased uterine wet weight. Of particular interest, ovx rats fed fermented soybeans showed increased uterine wet weights compared to control rats. Fermented soybean diet caused a significant increase in plasma 17-beta estradiol concentrations in young rats, and 17-beta estradiol levels were enhanced in ovx rats to match those of sham-operated ones. Significantly lower femoral BMD and BMC were observed in ovx rats compared to sham-operated controls, whereas bone areas did not differ statistically among the groups. In addition, BBS tended to be increased in ovx rats fed soybeans and fermented soybeans. CONCLUSIONS: Supplementation of fermented soybeans could have preventive and therapeutic effects against osteoporosis in postmenopausal women.
Animals ; Biological Availability ; Bone Density ; Caseins ; Decapitation ; Diet ; Estradiol ; Estrogens ; Female ; Fermentation ; Formaldehyde ; Glycosides ; Humans ; Isoflavones ; Menopause ; Metabolism ; Models, Animal ; Osteoporosis ; Ovariectomy ; Phytoestrogens ; Plasma ; Rats* ; Rats, Sprague-Dawley ; Receptors, Estrogen ; Rhizopus* ; Soybeans* ; Weight Gain ; Weights and Measures

OBJECTIVETo establish an appropriate neonatal rat model of necrotizing enterocolitis (NEC) and to investigate the protective effects of glycomacropeptide (GMP) on the gut from injury in neonatal rats with NEC.

METHODA total of 36 neonatal SD rats were randomly divided into 3 groups: NEC model group (Group M), NEC + GMP group (Group G) and normal control group (Group N), each group had 12 rats. All the neonatal rats were fed with breast milk in the first 3 days after birth. During the second 3 days after birth, the rats of Group N were still maternal breast-fed, but the rats of Group M and Group G were separated from their mothers and lived in incubator and began to be formula fed, and were subjected to cold exposure shortly after hypoxic-reoxygenation treatment. After being fed in such means for 6 days, all the neonatal rats were placed into the incubator and fasted for 24 hours. Then all the rats were sacrificed by cervical dislocation. Intestinal tissue located at the boundary of ileum and cecum was obtained for: (1) histological examination after HE staining, (2) TUNEL detection, (3) electron microscopic observation; and the tissue homogenate was obtained for checking TNF-α and IL-1β levels by ELISA and platelet activating factor (PAF) mRNA expression by quantitative fluorescence (QF)-PCR.

RESULT(1) The pathological scores of the 3 groups were 2.17 ± 0.83 (Group M), 0.92 ± 0.79 (Group G) and 0.17 ± 0.39 (Group N) separately. There was significant difference between Group M and Group G (H = 8.819, P = 0.003). (2) TNF-α levels of 3 groups were (41.94 ± 13.51) pg/ml (Group M), (31.69 ± 11.68) pg/ml (Group G) and (17.42 ± 7.18) pg/ml (Group N) separately, and TNF-α level in Group G was significantly lower than that of Group M (F = 3.959, P = 0.030). (3) IL-1β levels of 3 groups were (150.33 ± 36.41) pg/ml (Group M), (118.36 ± 33.00) pg/ml (Group G) and (28.44 ± 15.04) pg/ml (Group N) separately, and IL-1β level in Group G was lower than that of Group M (F = 5.080, P = 0.013). (4) Expression levels of intestinal PAF mRNA (2(-ΔΔCt) value): 3.01 ± 0.96 (Group M), 1.56 ± 0.29 (Group G), 1.01 ± 0.13 (Group N), the level of Group G was significantly lower than that of Group M (F = 25.251, P = 0.000). (5)Electron microscopy: Group N showed that its cell volume was mostly occupied by the nucleus, the structure was clear, nuclear membrane existed, suggesting the normal phase of cell; Group M showed that apoptotic body existed, suggesting that the advanced stage phase of apoptosis; Group G showed that condensed chromatin marginated around the nuclear envelope, nuclear pores expanded, suggesting the early phase of apoptosis. (6) The apoptosis rate of intestinal epithelial cells by TUNEL detection: 38.79 ± 9.79 (Group M), 29.54 ± 7.30 (Group G), 6.37 ± 1.96 (Group N); the apoptosis rate of intestinal epithelial cells of Group G was significantly lower than that of Group M (F = 6.888, P = 0.003).

CONCLUSIONGMP has protective effects on guts of neonatal rats with NEC, which may probably work by reducing TNF-α, IL-1β and PAF expression, inhibiting the apoptosis of intestinal epithelial cells and reducing intestinal tissue injury.

Animals ; Animals, Newborn ; Apoptosis ; Caseins ; pharmacology ; Cold Temperature ; Enterocolitis, Necrotizing ; drug therapy ; metabolism ; pathology ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Female ; Hypoxia ; complications ; Interleukin-1beta ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Intestines ; metabolism ; pathology ; Male ; Peptide Fragments ; pharmacology ; Platelet Activating Factor ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism

The aim of this study was to construct a mammary gland-specific expressional vector pBC1-hLF-Neo for Human Lactoferrin (hLF) gene and then investigate its expression in the mammary gland epithelium cells. The constructed vector contained the 6.2 kb long 5' flank regulation region including promoter, other elements and the 7.1 kb long 3' flank regulation region including transcriptional ending signal of a goat's beta-casein gene. A cassette of Neo gene was also inserted into the vector which gave a total length of 26.736 kb identified by restriction fragment analysis and partial DNA sequencing. The results revealed that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, we transfected the lined vector DNA into the dairy goat's mammary gland epithelium cells and C127 cells of a mouse's mammary epithelium by Lipofectamine. After selection with G418 for 8-10 days, G418-risistant clones were obtained. PCR analysis demonstrated that hLF gene cassette had been integrated into the genomic DNA of G418-risistant clones. After proliferation culture, the two kinds of transgenic cells were cultured in serum-free DMEM-F12 medium with prolactin, insulin and hydrocortisone- a medium capable of inducing recombinant hLF expression. RT-PCR, Western blotting and anti-bacteria bioactivity experiments demonstrated that the constructed mammary gland specific vector pBC1-hLF-Neo possessed the desirable bioactivity to efficiently express and could secrete hLF in both mammary gland cells and have the effect of E. coli proliferation inhibition. Paramount to everything, this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells.
Animals ; Base Sequence ; Breast Neoplasms ; metabolism ; pathology ; Caseins ; genetics ; Cell Line, Tumor ; DNA, Complementary ; biosynthesis ; genetics ; Epithelial Cells ; metabolism ; Female ; Genetic Vectors ; genetics ; Goats ; Humans ; Lactoferrin ; biosynthesis ; genetics ; Mammary Glands, Animal ; cytology ; metabolism ; Mice ; Molecular Sequence Data ; Mutagenesis, Insertional ; Promoter Regions, Genetic ; genetics

This study focused on the contamination mechanism and regeneration strategies of sulfopropyl ion exchange resin (SP Sepharose FF) during the separation of recombinant human lactoferrin from transgenic bovine milk. We analyzed primary constituents' contents in chromatorgraphic material and fractions. The results showed that the lipid in milk can clog the column or adhere to the resin through hydrophobic interaction, leading to an increase in column pressure. Some casein molecules were found to adsorb onto the resin through electrostatic interaction, therefore the adsorption capacity was decreased. There was no direct interaction between lactose and the resin in the chromatorgraphic process. Increased continuous chromatographic cycles and prolonged time interval between protein purification and column regeneration could enhance the undesirable interaction between the contaminants and resin, thus lowering the regeneration efficiency. NaOH was found to be effective in the removal of lipid and casein molecules from the column. Furthermore, normal microstructure and chromatographic performance of the ion exchanger was recovered after this cleaning procedure.
Adsorption ; Animals ; Animals, Genetically Modified ; genetics ; metabolism ; Caseins ; chemistry ; Cattle ; Chromatography, Ion Exchange ; methods ; Female ; Humans ; Ion Exchange Resins ; chemistry ; Lactoferrin ; biosynthesis ; genetics ; isolation & purification ; Mammary Glands, Animal ; metabolism ; Milk ; chemistry ; Milk Proteins ; isolation & purification ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sodium Hydroxide ; chemistry

To establish human beta-defensin-3 gene transgenic cell lines as competent donor cells for the production of transgenic animals using somatic cell nuclear transfer (SCNT). Firstly, we obtained human beta-defensin-3 by RT-PCR from human placenta, and subsequently inserted the fragment hBD into the corresponding site of the plasmid pBCP. Then we moved the combined fragment BCD (including 5' and 3' regulating region of beta-casein and hBD) into the corresponding site of the plasmid pEGFP-C1. Finally we successfully constructed mammary-specific expression vector pEBCD. We transected pEBCD into Holstein Fetal fibroblast cells by Lipofectamine TM-2000 and selected in medium with G418 for three to four weeks. We identified G418 resistant transfectants by PCR, RT-PCR and EGFP detection. Our results indicated that human beta-defensin-3 gene stably was integrated into the open region of the chromatin in G418 resistant fibroblast cells. Meanwhile we identified the expression of human beta-defensin-3 in the supernatant of stable transfected mammary epithelial cells by Western blotting. This study may provide competent transgenic donor cells for the production of transgenic animals by SCNT and improve the efficiency of transgenic cloning.
Animals ; Animals, Genetically Modified ; Caseins ; genetics ; Cattle ; Epithelial Cells ; metabolism ; Genes, erbB-1 ; genetics ; Genetic Vectors ; genetics ; Humans ; Mammary Glands, Animal ; cytology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; beta-Defensins ; biosynthesis ; genetics

This study was carried out to investigate the efficacy of sn-2 palmitic acid-fortified vegetable oil (Sn2PA) on calcium absorption and to confirm the synergistic effects of fructooligosaccharide on calcium absorption. Male SD rats were fed 6 kinds of casein based diets containing vegetable oil (control), sn-2 palmitic acid-fortified vegetable oil (Sn2PA) and Sn2PA with fructooligosaccharide(Sn2PAFO) in two levels of calcium (normal 0.5% and high 1.0%) for 3 weeks. Total lipids, cholesterol, triglyceride and calcium in blood were measured. Feces were collected using cages for 4 days. Serum concentrations of total lipids and calcium were not significantly different among groups. However, serum triglyceride was significantly decreased by fructooligosaccharide supplementation regardless of dietary calcium level. The lipid absorption was not significantly different among experimental groups. Calcium absorption was significantly higher in Sn2PAFO group than other groups. Calcium solubility of intestine was increased by sn-2 palmitic acid supplementation. These results suggest that sn-2 palmitic acid and fructooligosaccharide supplementation could be beneficial for baby foods including infant formula, with regard to increasing absorption of calcium by more soluble calcium in the small intestinal content.
Absorption ; Animals ; Calcium ; Calcium, Dietary ; Caseins ; Cholesterol ; Diet ; Feces ; Gastrointestinal Contents ; Humans ; Infant Formula ; Intestines ; Lipid Metabolism ; Male ; Oligosaccharides ; Palmitic Acid ; Rats ; Solubility ; Vegetables

This study was conducted to investigate the effect of soy protein hydrolyzate on lipid metabolism and antioxidant activity in the rat. Thirty-eight male rats of Sprague-Dawley strain were divided into five groups: casein, isolated soy protein (ISP), seoritae protein hydrolyzate (SH), soluble soy protein hydrolyzate (SS), and insoluble soy protein hydrolyzate (IS). The control diet (casein group) contained 20% casein protein and experimental diet contained 10% casein and 10% isolated soy-protein or soy-protein hydrolyzate. Fecal lipid content was increased and lipid apparent absorption rate was decreased significantly by the ISP group at the first week of experimental period. Blood triglyceride, total cholesterol, LDL-cholesterol and atherogenic index (AI) were decreased by soy protein hydrolyzate groups than casein group. Liver total lipid, triglyceride and cholesterol were not different among groups, but showed decreasing tendencies in soyprotein hydrolyzate groups. The lipid lowering effect was prominent in the IS group among soy protein hydrolyzate groups. Total antioxidant activity showed increasing tendency in the seoritae hydrolyzate group. Liver superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activities also showed higher tendencies in the seoritae hydrolyzate group than other groups. In conclusion, insoluble soyprotein hydrolyzate was more effective in lowering body lipids and seoritae hydrolyzate had higher antioxidant capacity among soy protein hydrolyzates.
Absorption ; Animals ; Caseins ; Catalase ; Cholesterol ; Diet ; Glutathione Peroxidase ; Humans ; Lipid Metabolism ; Liver ; Male ; Rats ; Soybean Proteins ; Sprains and Strains ; Superoxide Dismutase

A simple method of trypsin/collagenase I alternative digestion and iterative culture flask adherence to discard fibroblasts for bovine mammary cell culture was established in this study. By immunohistochemistry, flow cytometry, western blot, Electron microscopy analysis, the characteristics of bovine mammary cells were investigated in vitro. Effect of hyperthermia on the cell ultrastructures was also observed. The results showed that the mammary cells were diploid epithelia with intact 30 pairs chromatins, which could secrete alpha-casein into the medium. After exposed to hyperthermia, the cell condensed chromatin like crescent on the nuclei verges, mitochondria occurred expansion and vacuolization, and apoptotic bodies appeared, which suggested that heat stress could induce apoptosis of the mammary epithelia.
Animals ; Apoptosis ; Blotting, Western ; Caseins ; metabolism ; Cattle ; Cell Line ; Cell Nucleus ; ultrastructure ; Cells, Cultured ; Chromatin ; ultrastructure ; Epithelial Cells ; cytology ; metabolism ; ultrastructure ; Female ; Flow Cytometry ; Hot Temperature ; Immunohistochemistry ; Mammary Glands, Animal ; cytology ; metabolism ; Microscopy, Electron, Transmission ; Mitochondria ; ultrastructure ; Vimentin ; metabolism

An important related question is whether arginine has influence bone metabolism. The effect of arginine supplements on bone markers and related hormones were studied in young female Sprague-Dawley rats fed either an arginine supplemented diet or control diet. Twenty four rats (body weight 83 +/- 5 g) were randomly assigned to one of two groups, consuming casein or casein with supplemented arginine diet. All rats were fed on experimental diet and deionized water ad libitum for 9 weeks. Bone formation was measured by serum osteocalcin and alkaline phosphatase (ALP) concentrations. And bone resorption rate was measured by deoxypyridinoline (DPD) crosslinks immunoassay and corrected for creatinine. Serum osteocalcin, growth hormone, estrogen, insulin-like growth factor-1 (IGF-1), parathyroid hormone (PTH) and calcitonin were analyzed using radioimmunoassay kits. The weight gain and mean food intake were not affected regardless of diets. The rats fed arginine-supplemented diet had not significantly different in ALP, osteocalcin, crosslinks value, PTH, estradiol, and IGF-1 compared to those fed casein diet group. The arginine-supplemented group had significantly higher growth hormone and calcitonin than casein group. This study suggests that arginine is beneficial for bone formation in growing female rats. Therefore exposure to diet which rich in arginine early in life may have benefits for bone formation and osteoporosis prevention.
Alkaline Phosphatase ; Animals ; Arginine* ; Bone Resorption ; Calcitonin ; Caseins ; Creatinine ; Diet ; Eating ; Estradiol ; Estrogens ; Female* ; Growth Hormone ; Humans ; Immunoassay ; Insulin-Like Growth Factor I ; Metabolism ; Osteocalcin ; Osteogenesis ; Osteoporosis ; Parathyroid Hormone ; Radioimmunoassay ; Rats* ; Rats, Sprague-Dawley ; Water ; Weight Gain

This study was performed to investigate the effect of chongkukjang intake on lipid metabolism and liver function in ethanol consumed rats. Twenty one Sprague-Dawley male rats aging 4 weeks old were used as experimental animals, which were divided into three dietary groups: casein diet (CA), soybean diet (SB) and chongkukjang diet (CJ). Alcohol was consumed with water as 25% (v/v) ethanol solution. After 4 weeks of experimental period, rats were sacrificed to get blood and liver samples for analysis of lipids, lipid peroxides, antioxidative enzymes and biochemical indices of liver function. The mean body weight, food intake and liver index were not significantly different among three groups. Serum level of total lipid, triglyceride and total cholesterol of chongkukjang diet group was the lowest among three groups although the difference was not significant. HDL-cholesterol level was significantly (p < 0.05) higher in chongkukjang diet group than that of casein diet group. LDL-cholesterol level of chongkukjang and soybean diet group was significantly (p < 0.05) lower than that of casein diet group respectively. Liver TBARS of chongkukjang and soybean diet group was significantly (p < 0.05) lower than that of casein diet group respectively. The superoxide dismutase activity of chongkukjang diet group was significantly (p < 0.05) higher than that of casein diet group. Catalase activity was not significantly different among three groups. As indices of liver function, glutamic oxaloacetic transminase (GOT), glutamic pyruvic transminase (GPT), gamma-glutamyl transpeptidase (gamma-GTP) and lactate dehydrogenase (LDH) were not significantly different among three groups. Serum alcohol concentration and activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were not significantly different among three groups. The chongkukjang diet seems to give a beneficial effect for improving lipid metabolism by increasing HDLcholesterol level and SOD activity while reducing liver TBARS level. However, effect on liver function has to be investigated further.
Aging ; Alcohol Dehydrogenase ; Aldehyde Dehydrogenase ; Animals ; Body Weight ; Caseins ; Catalase ; Cholesterol ; Diet ; Eating ; Ethanol* ; gamma-Glutamyltransferase ; Humans ; L-Lactate Dehydrogenase ; Lipid Metabolism* ; Lipid Peroxides ; Liver* ; Male ; Rats* ; Rats, Sprague-Dawley ; Soybeans ; Superoxide Dismutase ; Thiobarbituric Acid Reactive Substances ; Triglycerides ; Water