Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein

The lactating mammary gland utilizes free plasma amino acids as well as those derived by hydrolysis from circulating short-chain peptides for protein synthesis. Apart from the major route of amino acid nitrogen delivery to the gland by the various transporters for free amino acids, it has been suggested that dipeptides may also be taken up in intact form to serve as a source of amino acids. The identification of peptide transporters in the mammary gland may therefore provide new insights into protein metabolism and secretion by the gland. The expression and distribution of the high-affinity type proton-coupled peptide transporter PEPT2 were investigated in rat lactating mammary gland as well as in human epithelial cells derived from breast milk. By use of RT-PCR, PEPT2 mRNA was detected in rat mammary gland extracts and human milk epithelial cells. The expression pattern of PEPT2 mRNA revealed a localization in epithelial cells of ducts and glands by nonisotopic high resolution in situ hybridization. In addition, immunohistochemistry was carried out and showed transporter immunoreactivity in the same epithelial cells of the glands and ducts. In addition, two-electrode voltage clamp recordings using PEPT2-expressing Xenopus laevis oocytes demonstrated positive inward currents induced by selected dipeptides that may play a role in aminonitrogen handling in mammalian mammary gland. Taken together, these data suggest that PEPT2 is expressed in mammary gland epithelia, in which it may contribute to the reuptake of short-chain peptides derived from hydrolysis of milk proteins secreted into the lumen. Whereas PEPT2 also transports a variety of drugs, such as selected beta-lactams, angiotensin-converting enzyme inhibitors, and antiviral and anticancer metabolites, their efficient reabsorption via PEPT2 may reduce the burden of xenobiotics in milk.     

A quantitative assessment of the contribution of individual plasma amino acids to the synthesis of milk proteins by the goat mammary gland

1. Arteriovenous differences of plasma free amino acids across the lactating mammary glands of six goats have been measured. 2. In four experiments, measurements of blood flow, amino acid arteriovenous differences, milk yield and milk nitrogen showed that the uptake of nitrogen in the form of amino acids was sufficient to provide all the nitrogen of the milk proteins synthesized in the mammary gland. 3. In the same four experiments the uptake from the plasma and output into the milk of individual amino acids per unit time were compared. The uptakes of essential amino acids and glutamic acid were approximately equal to the corresponding output figures. The uptake of serine was consistently less than the output, and the uptake of other non-essential amino acids was very variable, in some experiments being approximately equal to the output figures and in others being considerably less. 4. As in cows, there was an uptake of ornithine in all experiments, though ornithine is absent from milk. In goats, though not in cows, the uptake of arginine was consistently greatly in excess of the requirement for arginine residues in milk protein. 5. The possible significance of the uptakes of arginine and ornithine for the synthesis of serine and other non-essential amino acids in the mammary gland is discussed. 6. The importance of clamping the external pudic vein, when sampling mammary venous blood from the caudal superficial epigastric vein, is indicated.     

Bile salt-stimulated lipase (BSSL) distribution in rat, mouse and transgenic mouse expressing human BSSL

 In some species, including man and mouse, bile salt-stimulated lipase (BSSL) in milk catalyzes the hydrolysis of triacylglycerides into glycerol and free fatty acids, a reaction that is of particular importance during suckling. The enzyme is also secreted by the pancreas (referred to as carboxyl-ester hydrolase, CEH). We wished to localize sources and storage sites for BSSL/CEH in rats, in wild-type mice, and in transgenic mice producing recombinant human BSSL in milk. Immunoreactivity against several BSSL fragments was strong in the pancreatic acinar cells and moderate in the absorptive cells of the small intestine and in salivary duct cells of the mice, as well as in rats. Sections from lactating mammary glands of mouse, but not rat, also showed immunoreactivity for BSSL; the signal was strongest in the transgenic mice. Radioactive riboprobes for BSSL mRNA hybridized on sections of rat and mouse pancreatic acinar cells, and mouse mammary glands (both wild-type and transgenic). Using RT-PCR, it was possible to amplify BSSL mRNA from wild-type mouse pancreas and mammary gland, from rat submandibular glands, and, in a few cases, from rat liver. In transgenic mice, the BSSL mRNA was highly expressed only in lactating mammary gland, but could be detected in a few other organs as well. Accepted: 31 March 1998     

Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene.

To extend our understanding of the organization and expression of the mouse mammary tumor virus genome, we determined the nucleotide sequence of large regions of a cloned mouse mammary tumor virus strain C3H provirus that appears to be a DNA copy of env mRNA. In conjunction with analysis of several additional clones of integrated and unintegrated mouse mammary tumor virus DNAs, we came to the following conclusions: (i) the mRNA for env is generated by splicing mechanisms that recognize conventional eucaryotic signals at donor and acceptor sites with a leader of at least 289 bases in length; (ii) the first of three possible initiation codons for translation of env follows the splice junction by a single nucleotide and produces a signal peptide of 98 amino acids; (iii) the amino terminal sequence of the major virion glycoprotein gp52env is confirmed by nucleotide sequencing and is encoded by a sequence beginning 584 nucleotides from the 5' end of env mRNA; (iv) the final 17 amino acids at the carboxyl terminus of the primary product of env are encoded within the long terminal repeat by the 51 bases at the 5' end of the U3 domain; and (v) bases 2 through 4 at the 5' end of the long terminal repeat constitute an initiation codon that commences an open reading frame capable of directing the synthesis of a 36-kilodalton protein.     

Lysine tRNA is the predominant tRNA in murine mammary tumor virus

The method of aminoacylation and subsequent identification of the esterified amino acids was used to characterize the transfer RNAs in murine mammary tumor virus. Lysine tRNA was the major tRNA in both “free” 4S RNA and “7OS-associated” 4S RNA in virus derived from either tissue culture or mouse milk.     

A static model to analyze carbon and nitrogen partitioning in the mammary gland of lactating sows

Quantitative estimates of mammary nutrient inputs, outputs and metabolism in sows are scarce, despite being critical elements to identify parameters controlling milk synthesis central for the feeding of lactating sows. The objective of this study was to quantify the mammary gland input and output of nutrients as well as the intramammary partitioning of carbon and nitrogen with the purpose to identify mechanisms controlling mammary nutrient inputs, metabolism and milk production in lactating sows. A data set was assembled by integration of results from four studies. The data set included data on litter performance, mammary arterial-venous concentration differences (AV-difference) of energy metabolites and amino acids, and the contents of lactose, fat and amino acids in milk. Milk yield was estimated based on average litter size and litter gain, and mammary plasma flow (MPF) was estimated using the sum of phenylalanine and tyrosine as internal flow markers. The yield and composition of milk were used to estimate mammary nutrient output in milk, and MPF and AV-difference were used to estimate net mammary input of carbon and nitrogen and output of CO₂. Carbon and nitrogen used for the synthesis of lactose, fat and protein in milk and CO₂-yielding processes were represented in a static nutrient partitioning model. The origin of mammary CO₂ output was calculated using theoretical estimates of carbon released in processes supporting mammary synthesis of de novo fat, protein and lactose in milk, mammary tissue protein turnover and transport of glucose and amino acids. Results indicated that total input of carbon from glucose and lactate was partitioned into lactose (36%), fat (31%) and CO₂-yielding processes (34%). Theoretical CO₂ estimates indicated that de novo fat synthesis, milk protein synthesis and mammary tissue protein turnover were the main processes related to mammary CO₂ production. More than 90% of mammary gland amino acid input was used for milk protein. The quadratic relationship between AV-difference and mammary input of essential amino acids indicated that both changes in AV-difference and MPF contributed to the regulation of mammary input of essential amino acids. The impact of the arterial supply of amino acids on mammary input may be greater for the branched-chain amino acids, arginine and phenylalanine than for other essential amino acids. In conclusion, relationships between input and output parameters indicate that AV-difference and MPF regulate mammary nutrient input to match the supply and demand of nutrients for the mammary gland.     

Free Amino Acids and Phosphorylethanolamine in Milk Whey of Cow

Free amino acids in the milk of cow were investigated in comparison with those in the plasma. The concentrations of most free amino acids in the milk except for a few amino acid were lower than those in the plasma. It appears that the percentage of each amino acid in the milk against the corresponding amino acid in the plasma is the reflexion of casein synthesis in the mammary gland. Nutritional alteration influenced on the level of some amino acids in the milk. Free phosphorylserine, glycerylphosphorylethanolamine, and phosphorylethanolamine were observed in the milk. Phosphorylethanolamine was present in significantly high concentration in one animal as control, whereas was almost absent in another animal as experimental.     

棉铃虫感染中华卵索线虫后血淋巴游离氨基酸含量的变化

中华卵索线虫Ovomermis sinensis感染棉铃虫Helicoverpa armigera 1天后,棉铃虫幼虫血淋巴中游离氨基酸总量大幅度下降,各种游离氨基酸的含量也是如此变化。感染2～4天后的棉铃虫血淋巴中游离氨基酸总量变化不大,各种游离氨基酸的含量有升有降。感染5～6天后的棉铃虫血淋巴中游离氨基酸总量和各种氨基酸的含量都急剧上升。研究表明,中华卵索线虫寄生棉铃虫时,棉铃虫血淋巴中游离氨基酸含量的变化朝着有利于线虫生长发育的方向进行。     

Expression of human lysozyme mRNA in the mammary gland of transgenic mice

Owing to its inherent antimicrobial effect and positive charge, the expression of human lysozyme in bovine milk could be beneficial by altering the overal microbial level and the functional and physical properties of the milk. We have used transgenic mice as model systems to evaluate the expression of human lysozyme containing fusion gene constructs in the mammary gland. Expression of human lysozyme was targeted to the mammary gland by using the 5 promoter elements of either the bovine (line B mice) or _s1 (line H mice) casein genes coupled to the cDNA for human lysozyme. Expression of human lysozyme mRNA was not found in mammary tissue from any of line B mice. Tissues were analysed from six lines of H mice and two, H6 and H5, were found to express human lysozyme mRNA in the mammary gland at 42% and 116%, respectively, of the levels of the endogenous mouse whey acidic protein gene. At peak lactation, female mice homozygous for the H5 and H6 transgene have approximately twice the amount of mRNA encoding human lysozyme as hemizygous animals. Expression levels of human lysozyme mRNA in the mammary gland at time points representing late pregnancy, early, peak and late lactation corresponded to the profile of casein gene expression. Human lysozyme mRNA expression was not observed in transgenic males, virgin females or in the kidney, liver, spleen or brain of lactating females. A very low level of expression of human lysozyme mRNA was observed in the salivary gland of line H5.     

Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland.

The mRNA sequence for bovine lactoferrin expressed in the mammary gland was determined by sequencing three over lapping cDNA clones and by direct sequencing of the mRNA. The mRNA (2351 bases) codes for a 708 amino acid protein with a 19 amino acid signal peptide immediately preceding a sequence identical to the N-terminal 40 amino acids reported for bovine lactoferrin. A putative destabilizing sequence (AUUUA) was identified in the 3'-untranslated region. The nucleic acid sequence and deduced amino acid sequence are highly homologous with other transferrin family members. Lactoferrin mRNA concentrations in bovine mammary tissue were quite low two days before parturition and during lactation but were high three days after the cessation of milking, a sharp contrast from the pattern of regulation of the other milk proteins.     

In Vitro System for Production of Mouse Mammary Tumor Virus

An in vitro system for production, purification, and concentration of mouse mammary tumor virus is described. Monolayer cultures of C(3)H mouse mammary tumor cells propagated at 34 C in roller bottles in the presence of dexamethasone, a glucocorticoid hormone, release B-type particles which possess ribonucleic acid and a ribonucleic acid-dependent deoxyribonucleic acid polymerase. One thousandfold concentration by ultracentrifugation with subsequent gradient fractionation yielded > 7 x 10(10) particles per ml in the 1.16- to 1.18-g/ml region. Mouse mammary tumor virus produced in this system was free of detectable C-type virus.     

大熊猫乳汁中富含游离精氨酸

采用高效液相色谱法测定了8只圈养大熊猫20个乳样中游离氨基酸的含量。结果显示:大熊猫初乳和常乳中均含有丰富的游离精氨酸,并且是含量最高的游离氨基酸;泌乳2-10d的大熊猫初乳中总游离氨基酸含量约为82mg/100ml,其中游离精氨酸平均含量达61mg/100ml,常乳中游离精氨酸含量约为54mg/100ml,均明显高于人、牛和藏绵羊乳;游离精氨酸在大熊猫干乳期乳腺分泌物中含量显著下降。推测乳中高水平的游离精氨酸在大熊猫幼仔生长发育中可能发挥重要作用[动物学报52(2):309-315,2006]。     

EPR characterization of free radical intermediates formed during ultrasound exposure of cell culture media

Free radicals and/or hydrogen peroxide produced by exposure of cells to ultrasound are potentially cytotoxic and mutagenic. The formation and type of free radical species can be substantially modulated by the chemical composition of the media in which the ultrasound exposures of cells are carried out. In the current study, we examined the free radical intermediates formed during ultrasound exposure of a typical cell culture medium (RPMI-1640); the dominant free radicals that were identified by spin trapping were derived from the hydrophobic amino acids Trp, Leu, and Phe, and were formed by hydrogen abstraction from these amino acids. Compared to exposures in phosphate-buffered saline, the yield of *OH radicals and H2O2 was significantly reduced in the cell culture medium, glucose (the main organic component in the medium), and the hydrophobic amino acids (Trp, Phe, Tyr, Leu, Val, Met) being chiefly responsible for this effect. In contrast, other nonhydrophobic amino acids did not contribute significantly to the *OH or H2O2 decrease. These findings are consistent with the accumulation of hydrophobic solutes at the liquid-gas interface of the collapsing cavitation bubbles resulting in increased efficiency of radical scavenging.     

Cloning and analysis of cDNA encoding bovine butyrophilin, an apical glycoprotein expressed in mammary tissue and secreted in association with the milk-fat globule membrane during lactation

Butyrophilin is a glycoprotein expressed on the apical surfaces of secretory cells in lactating mammary tissue, which may function in the secretion of milk-fat droplets (Franke, W. W., Heid, H. W., Grund, C., Winter, S., Freudenstein, C., Schmid, E., Jarasch, E.-D., and Keenan, T. W. (1981) J. Cell Biol. 89, 485-494). A cDNA clone encoding bovine butyrophilin was isolated and the primary structure of the protein deduced from the DNA sequence. Bovine butyrophilin contains 526 amino acids with a putative signal peptide of 26 amino acids. Hydropathy analysis predicts the existence of a single membrane-spanning region with the amino terminus facing the exoplasmic space. Butyrophilin cDNA hybridized to mRNA of 2.9 kilobases in bovine mammary tissue taken from pregnant or lactating animals, but specific mRNA was not detected in a 2-year-old virgin cow. The amount of message was maximal in lactating tissue. Butyrophilin mRNA could not be detected in bovine heart, intestine, kidney, liver, ovary, or uterus. A search of the GenBank and EMBL data banks showed closest homology was between the C termini of butyrophilin and "ret finger protein" (Takahashi, M., Inaguma, Y., Hiai, H., and Hirose, F. (1988) Mol. Cell. Biol. 8, 1853-1856). The ret-finger protein gene is expressed in a variety of tumor cell lines, mouse testis and embryonic tissue, which like the mammary gland undergo periods of rapid cell division and development. The possible significance of this homology and the possible function of butyrophilin in milk-lipid secretion are discussed.     

Cloning of a novel gene specifically expressed in clonal mouse chondroprogenitor-like EC cells, ATDC5

We cloned a full-length cDNA encoding a novel mouse protein, A-C2, by differential display method using mouse embryonic fibroblast C3H10T1/2 cells and mouse chondroprogenitor-like EC cells, ATDC5. The deduced amino acid sequence of A-C2 consisted of 106 amino acids with no significant homology to the sequences previously reported. Northern blot analysis showed two major bands of 2.1 and 1.8 kb sizes. Expression of A-C2 mRNA was exclusive to ATDC5 cells at their undifferentiated stage. None of ATDC5 cells at their differentiated stage and adult mice tissues examined expressed A-C2 gene.     

Studies on the mode of uptake of blood triglycerides by the mammary gland of the lactating goat. The uptake and incorporation into milk fat and mammary lymph of labelled glycerol, fatty acids and triglycerides

1. The mode of uptake of the precursors of milk fat by the mammary gland of the lactating goat has been examined by infusing radioactive fatty acids, glucerol or doubly labelled triglycerides into the mammary artery or jugular vein of animals surgically prepared to permit samples of arterial and venous blood to be withdrawn without disturbance to the animal. 2. Acetate was taken up by the mammary gland and incorporated into milk fat. The decrease in the specific radioactivity of blood acetate across the gland was evidence of acetate production, but there was no significant release of labelled lipid from the mammary gland. 3. When labelled long-chain fatty acids or glycerol were infused into the lactating goat, there was extensive transfer of radioactivity into milk in spite of the absence of net uptake of substrate by the mammary gland. The decrease in the specific radioactivity of each substrate across the mammary gland, however, showed that both fatty acids and glycerol were simultaneously taken up and released by mammary tissue. 4. The infusion of chylomicra and triglyceride emulsions labelled with (3)H and (14)C revealed that both glycerol and fatty acids were released during triglyceride uptake by mammary tissue. Changes in the (3)H/(14)C ratio during the transfer of triglyceride from blood into milk showed that at least 80% of the triglyceride was hydrolysed during uptake, but the potential re-utilization of both products of hydrolysis for triglyceride synthesis in mammary tissue implied that only a minimum value could be obtained from the change in the ratio. 5. The time-course of the transfer of (3)H and (14)C into milk and lymph were closely similar after the infusion of [2-(3)H]glycerol tri[1-(14)C]oleate or of a mixture of [2-(3)H]glycerol and [1-(14)C]oleate. 6. The results were consistent with the hypothesis that plasma triglycerides are extensively or completely hydrolysed during mammary uptake.