Role of glucocorticoids and progesterone in the development of rough endoplasmic reticulum involved in casein biosynthesis.

Hydrocortisone acetate injected into pseudopregnant rabbits induced casein synthesis and a parallel accumulation of casein mRNA. These effects were not accompanied by any enrichment of total RNA in the mammary cell. Hydrocortisone acetate did not favour the attachment of polysomes to endoplasmic reticulum. Casein mRNA concentration was enhanced in free and membrane-bound polysomes. After long treatments, the concentration of casein mRNA reached a plateau in membrane bound polysomes whereas it continued to be accumulated in free polysomes, suggesting that a substantial part of casein synthesis is then carried out by free polysomes. Progesterone injected with high doses of prolactin was unable to prevent the stimulatory action of prolactin on the synthesis of casein, the accumulation of casein mRNA and mammary gland growth, as judged by DNA content. By contrast, the increase in the total RNA content of mammary gland was still significantly reduced by progesterone. In addition, progesterone inhibited almost completely the formation of membrane-bound polysomes and the anchorage of casein mRNA to endoplasmic reticulum. From these data, it was concluded that the formation of the endoplasmic reticulum is not a prerequisite for the initiation of casein synthesis. Glucocorticoids do not play a major role in the formation of the endoplasmic reticulum and the Golai apparatus and in the binding of casein synthesizing polysomes to membranes. Progesteronne is capable of inhibiting preferentially and gradually the stimulation of cellular functions requiring the most potent prolactin stimulation.     

Simultaneous occurrence of pregnancylike lobuloalveolar morphogenesis and casein-gene expression in a culture of the whole mammary gland

Summary Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 μg/ml), aldosterone (1 μg/ml), growth hormone (5 μg/ml), cortisol (5 μg/ml), and prolactin (80 ng/ml, present as a contaminant in 5 μg/ml growth hormone). The alveolar growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA probe (cDNA_csn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum mammary gland in vivo. This work was supported by Department of Health, Education and Welfare Grants CA11058 and CA25304 from the National Cancer Institute.     

An essential role for glucocorticoid in casein gene expression in rat mammary explants

It is demonstrated that the accumulation of 42 K casein mRNA in mammary tissue from adrenalectomized, virgin rats is almost 20x higher in the presence of exogenous hydrocortisone than in its absence. Accumulation of 25 K casein mRNA in this tissue is totally dependent on the steroid. The results indicate a much greater dependency on hydrocortisone than was appreciated previously, and also show that this dependency does not reflect a loss of cell viability in the absence of the steroid.     

Characterization of tryptic casein phosphopeptides prepared under industrially relevant conditions

Anticariogenic casein phosphopeptides (ACPP) contain the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- and have commercial potential as toothpaste, mouthwash, and food additives for the prevention of dental caries. In an approach to develop a commercial-scale process for the production of ACPP we have comprehensively characterized casein phosphopeptides (CPP) produced under industrially relevant conditions. Sodium caseinate (10% w/v) was hydrolyzed by Novo trypsin (commercial grade) at 50 degrees C for 2 h and CPP were purified from the acid clarified hydrolysate by a single-step selective precipitation procedure involving Ca(2+) (20 mol/mol casein) and ethanol (50% v/v) at pH 4.6 or 8.0. The individual peptides of the CPP preparations were purified by reversed-phase high-performance liquid chromatography (HPLC) and then identified by amino acid composition and sequence analyses. The yield of the pH 8.0 precipitate (13.85 +/- 0.48 wt % of the caseinate) was slightly higher than that of the pH 4.6 precipitate (11.04 +/- 0.30 wt % of the caseinate). However, the pH 4.6 precipitate contained predominantly (86.4 mol %) ACPP cluster peptides with small amounts of the diphosphorylated peptides (13.6 mol %), alpha(s1)(43-58) and alpha(s2)(126-136). In the pH 8.0 precipitate the cluster peptides represented a smaller proportion of the total peptides (61.9 mol %) due to increased recoveries of the diphosphorylated peptides (24.4 mol %) as well as the additional recovery of the monophosphorylated peptide beta(33-48) (13.7 mol %) indicating increased cross-linking by Ca(2+) at the higher pH. The recovery of the ACPP from the original caseinate was similar for both the pH 4.6 and 8.0 precipitates. Slight chymotryptic activity was detected in the industrial-grade enzyme, resulting in minor truncation of some peptides. Also some deamidation and methionine oxidation of one peptide, alpha(s1)(59-79), were detected. In conclusion, ACPP can be produced under industrially relevant conditions with only minor modifications such as slight truncation, deamidation, and methionine oxidation. However, in order to prepare casein phosphopeptides predominantly containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, the single-step selective precipitation with Ca(2+)/ethanol should be performed at pH 4.6 rather than pH 8.0. (c) 1995 John Wiley & Sons, Inc.     

Caseins of various origins and biologically active casein peptides and oligosaccharides: Structural and physiological aspects

Summary The first part of the present review is focused on structural aspects concerning the so far studied casein fractions of various origins: they are compared to the four classical major bovine caseins (_sl-, _s2- - and ). The calcium-sensitive casein fractions are always phosphorylated whereas -caseins are glycosylated. The study of the casein genes showed that the calcium-sensitive caseins diverged from a common ancestral gene and during the evolution, intergenic and intragenic duplications occurred. The considerable conservation of the phosphorylation sites emphasizes the importance of phosphorylated residues for the function of caseins, i.e. the formation of micelles and the binding of Ca²⁺. In -caseins all the prosthetic sugar groups are linked by O-glycosidic linkages: their number varies from 0 to 5 in bovine -casein and up to 10 in human -casein. The structures of the known -casein carbohydate moieties are described. Finally the milk clotting process (interaction -casein/chymosin) is compared to the blood clotting process (interaction fibrinogen/thrombin): a large number of similarities could be noted between both clotting phenomena.The second part of the review is devoted to the study of short casein peptides endowed with various biological activities. Some of them behaved as immunomodulators or casomorphins or angiotensin I converting enzyme inhibitors; others demonstrated an effect on platelet functions. A strategic zone containing immunostimulating and opioid peptides could be located in cow and human -caseins. Furthermore bitter peptides, emulsifying peptides, calcium absorption enhancing peptides, chymosin-inhibiting peptides, have also been described and several further properties have been attributed to the -caseinoglycopeptide; two tetrasaccharides isolated from the latter possess blood group activities.In conclusion caseins, the main milk proteins, should not only be considered as a nutriment but as a possible source of biologically active components.If, in the future, some of the discussed active peptides cannot be characterized in vivo, they can all, nevertheless, be synthesized and used either as food additives or in pharmacology.     

体外法探究不同氨基酸形式酪蛋白水解物对猪小肠微生物的影响

【目的】通过体外法探究猪小肠不同肠段肠腔微生物和肠壁微生物对两种不同氨基酸形式的酪蛋白水解物的发酵特性。【方法】以生长猪的十二指肠、空肠、回肠肠腔食糜或者肠壁微生物为接种物,分别以酸水解酪蛋白(以游离氨基酸为主)和酶水解酪蛋白(以小肽为主)为底物,37°C厌氧培养发酵,于0、3、6、12 h采样,测定微生物蛋白(MCP)以及用real time-PCR进行菌群分析。【结果】(1)肠腔微生物发酵不同酪蛋白水解物:十二指肠和回肠酶水解酪蛋白组MCP的含量显著高于酸水解酪蛋白组(P0.05)。十二指肠酶水解酪蛋白组总菌、Firmicutes数量显著高于酸水解酪蛋白组(P0.05)。回肠发酵6 h后,酶水解酪蛋白组Escherichia coli和Firmicutes的数量显著高于酸水解酪蛋白组(P0.05);发酵12 h后,酶水解酪蛋白组总菌、Lactobacillus、E.coli的数量均显著高于酸水解酪蛋白组(P0.05)。(2)肠壁微生物发酵不同酪蛋白水解物:发酵12 h后,十二指肠和回肠酶水解酪蛋白组MCP含量显著高于酸水解酪蛋白组(P0.05)。十二指肠酶水解酪蛋白组Lactobacillus、Firmicutes数量分别极显著、显著高于酸水解酪蛋白组;回肠Firmicutes数量在酶水解酪蛋白组显著高于酸水解酪蛋白组(P0.05)。【结论】十二指肠和回肠的肠腔和肠壁微生物都能够利用小肽,且在一定程度上对小肽的利用更具优势。     

Relationship Between mRNA Stability and Length: An Old Question with a New Twist

The half-life of individual mRNA plays a central role in controlling the level of gene expression. However, the determinants of mRNA stability have not yet been well defined. Most previous studies suggest that mRNA length does not affect its stability. Here, we show significant negative correlations between mRNA length and stability in human and Escherichia coli, but not in Saccharomyces cerevisiae or Bacillus subtilis. This finding suggests the possibility that endonucleolytic attacks by RNA endonuclease and/or mechanical damage may strongly influence mRNA stability in both prokaryotes and eukaryotes.     

木瓜凝乳蛋白酶的酶学性质研究

以酪蛋白为底物,木瓜凝乳蛋白酶的最适反应温度为80℃(pH7.0),最适pH为3-5(37℃)。在pH为7.0、反应温度为37℃的条件下,木瓜凝乳蛋白酶的Km值为1.25g·L-1,Vmax为0.1 g·L-1min-1。低浓度的NaCl和Ca2+对木瓜凝乳蛋白酶有激活作用,盐酸胍对其有抑制作用。     

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels

Summary The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion. This investigation was supported by Grant No. CA 05388 awarded by the National Cancer Institute, DHEW, and by Cancer Research Funds of the University of California.     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Identification of osteopontin phosphorylation sites involved in bone remodeling and inhibition of pathological calcification

Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.     

Bioavailability of lead from various milk diets studied in a suckling rat model

The bioavailability of lead from various milk diets was studied in 14 day old suckling rats. Human milk, infant formula, cow's milk, rat milk and deionized water labeled with ²⁰³Pb were given to rat pups by gastric intubation. Animals were killed after 2 or 6 h and the radioactivity in the tissues was measured. At 2 h after administration the lead bioavailability, defined as lead uptake in the body, excluding the gastrointestinal tract, was 47% from water, 42% from human milk, 40% from infant formula, 31% from cow's milk and 11% from rat milk. After 6 h the bioavailability of lead was about 50% from water and human milk, 45% from infant formula and cow's milk, and 36% from rat milk. The blood lead levels in the pups reflected the total body uptake and were also correlated to the brain lead levels. Thus, rat pups given lead in human milk had approximately twice as high lead levels in blood and brain than pups given lead in rat milk. The intestinal absorption of lead was dependent on the milk diet given to the sucklings. In duodenum, the highest uptake of lead was found in rats given water or human milk, whereas in rats given rat or cow's milk the highest uptake of lead was found in ileum. The distribution of lead in cream, whey and casein fractions of the milk diets after in vitro labeling with ²⁰³Pb was also studied. The casein fraction in cow's and rat milk contained 90–96% of the total amount of lead in the diet. In infant formula and human milk, 77 and 56% lead was found in the casein fraction, respectively. The higher lead bioavailability observed in the suckling rat fed human milk than in those fed rat and cow's milk may partly be explained by a lower proportion of lead bound to casein in human milk.     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.     

Assessment of the efficiency of nitrogen utilization in the infant cebus monkey (Cebus albifrons) by nitrogen balance

Nitrogen (N) balance and growth were utilized to assess the efficiency of N utilization in the infant cebus monkey (Cebus albifrons). The efficiency of N utilization as calculated from N balance data was 35%. The efficiency of N utilization for growth was 37% as determined by weight change over a 28-day trial and by body composition data from the literature. These results indicate, therefore, that growth and N balance are comparable indicators of N utilization in these primates.     

脑益康对D-半乳糖和亚硝酸钠所致Alzheimer病小鼠模型的保护作用

目的:探讨脑益康(中药)对D-半乳糖(D-gal)和亚硝酸钠(NaNO2)所致阿尔茨海默病(AD)小鼠模型的保护作用.方法:采用D-gal和NaNO2腹腔注射建立AD小鼠模型.应用迷宫刺激器检测小鼠学习记忆能力,生化方法检测小鼠脑组织一氧化氮(NO)含量和单胺氧化酶-B(MAO-B)、谷胱甘肽过氧化物酶(GSH-PX)、Na -K -ATP酶及Ca2 -ATP酶活性; RT-PCR检测凋亡调控基因Bax、Bcl-2 mRNA表达情况.结果:脑益康(中药)能改善小鼠学习记忆能力,降低AD小鼠脑组织MAO-B活性,升高Na -K -ATP酶和Ca2 -ATP酶活性,抑制Bax mRNA的表达,上调Bcl-2 mRNA的表达.结论:脑益康(中药)对AD小鼠有一定保护作用,其机制可能与降低脑组织MAO-B活性,提高脑组织Na -K -ATP酶和Ca2 -ATP酶活性,调节Bcl-2 mRNA和Bax mRNA的表达,发挥抗氧化作用,减轻神经细胞损伤有关.     

Micellization of casein-graft-dextran copolymer prepared through Maillard reaction

Casein is almost insoluble at around pH 4.6, which is its isoelectric point (pI). Grafting copolymer, casein-g-dextran, was prepared through the Amadori rearrangement of the Maillard reaction. The copolymer has a reversible pH sensitive property: micellization at the pI of casein forming a casein core and dextran shell structure and dissociation when pH differs from the pI. The micelles produced at pH 4.6 have a spherical shape and their size is dependent on the Maillard reaction: reaction time, molar ratio of casein to dextran, and molecular weight of dextran used. Typically, the hydrodynamic diameter of the micelles is about 100 nm and the critical micelle concentration is about 10 mg/L. The micelles are very stable in aqueous solution and can be stored as lyophiled powder. The micelles are able to encapsulate hydrophobic compounds such as pyrene.     

The DHH1/RCKp54 family of helicases: An ancient family of proteins that promote translational silencing

Translational control is a vital aspect of gene expression. Message specific translational repressors have been known for decades. Recent evidence, however, suggests that a general machinery exists that dampens the translational capacity of the majority of mRNAs. This activity has been best ascribed to a conserved family of RNA helicases called the DHH1/RCKp54 family. The function of these helicases is to promote translational silencing. By transitioning mRNA into quiescence, DHH1/RCKp54 helicases promote either mRNA destruction or storage. In this review we describe the known roles of these helicases and propose a mechanistic model to explain their mode of action. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.