Regulation of protein synthesis in lactating rat mammary tissue by cell volume

The effect of changing cell volume on rat mammary protein synthesis has been examined. Cell swelling, induced by a hyposmotic challenge, markedly increased the incorporation of radiolabelled amino acids (leucine and methionine) into trichloroacetic acid (TCA)-precipitable material: reducing the osmolality by 47% increased leucine and methionine incorporation into mammary protein by 147 and 126% respectively. Conversely, cell shrinking, induced by a hyperosmotic shock, almost abolished the incorporation of radiolabelled amino acids into mammary protein: increasing the osmolality by 70% reduced leucine and methionine incorporation into mammary protein by 86 and 93% respectively. The effects of cell swelling and shrinking were fully reversible. Volume-sensitive mammary tissue protein synthesis was dependent upon the extent of the osmotic challenge. Isosmotic swelling of mammary tissue, using a buffer containing urea (160 mM), increased the incorporation of radiolabelled leucine into TCA-precipitable material by 106%. Swelling-induced mammary protein synthesis was dependent upon calcium: removing extracellular calcium together with the addition of EGTA markedly reduced volume-activated protein synthesis. Cell swelling-induced protein synthesis was inhibited by the Ca(2+) ATPase blocker thapsigargin suggesting that volume-sensitive protein synthesis is dependent upon luminal calcium.     

Influence of torpor on milk protein composition and secretion in lactating bats.

In the pipistrelle bat (Pipistrellus pipistrellus), the metabolic load of lactation is not met to any significant extent by increased food intake or mobilization of body reserves, and aerial foraging accounts for most of the animal's energy expenditure even during lactation. Energy conservation must, therefore, play a critical role in maintaining lactation. The principal mechanism for energy conservation appears to be the bat's ability to enter torpor, but this may itself interrupt milk synthesis and secretion unless the pipistrelle mammary gland is adapted to counteract its effect. The effect of torpor on mammary tissue function was studied in mammary tissue explant cultures prepared in weeks 1-3 of lactation, when milk water yield was 0.20, 0.88, and 0.30 mL/d respectively. Protein synthesis measured by incorporation of radiolabeled amino acids was 44% lower (P < 0.001) in bat tissue explants cultured at ambient temperature (22 degrees C) compared with 37 degrees C. The reduction was similar to that observed in mouse mammary tissue (57%) and was unaffected by stage of lactation. Analysis of explant protein after [35S]methionine labelling showed the majority of proteins synthesised in culture to be milk proteins; it also demonstrated that the decrease in protein synthesis at ambient temperature was a general phenomenon: synthesis of both secretory and intracellular mammary proteins was reduced at the lower culture temperature. The results suggest that bat mammary tissue has no mechanism to counteract the effect of reduced body temperature and that periods of lactational torpor are likely to cause a pronounced diurnal variation in the rate of milk secretion.     

DEAD-box helicase 6 (DDX6) is a new negative regulator for milk synthesis and proliferation of bovine mammary epithelial cells

Milk synthesis of bovine mammary gland is a complex biological process that is regulated by hormones and nutrients, but the mechanism of these regulations still needs further research. DEAD-box helicase 6 (DDX6) is an important member of the RNA helicase family, involved in the regulation of mRNA storage and translation in different systems, but its physiological role and mechanism are largely unclear. In this study, we describe DDX6 as a potentially novel negative regulator for milk synthesis and proliferation of bovine mammary epithelial cells (BMECs). Treatment of BMECs with amino acids (methionine or leucine) or hormones (estrogen or prolactin) decreased the expression of DDX6. DDX6 expression was lower in mammary tissues of lactation period than in mammary tissues of puberty and dry period. Notably, overexpressing DDX6 in BMECs significantly decreased milk synthesis, cell proliferation, and protein levels of p-mTOR, SREBP-1c, and cyclin D1, while inhibiting DDX6 had the opposite effect. Taken together, these results reveal that DDX6 is a new negative regulator to control milk synthesis and proliferation of BMECs.     

Methionine protects against hyperthermia-induced cell injury in cultured bovine mammary epithelial cells

The aim of this study was to investigate the effects of methionine on cell proliferation, antioxidant activity, apoptosis, the expression levels of related genes (HSF-1, HSP70, Bax and Bcl-2) and the expression levels of protein (HSP70) in mammary epithelial cells, after heat treatment. Methionine (60 mg/L) increased the viability and attenuated morphological damage in hyperthermia-treated bovine mammary epithelial cells (BMECs). Additionally, methionine significantly reduced lactate dehydrogenase leakage, malondialdehyde formation, nitric oxide, and nitric oxide synthase activity. Superoxide dismutase, catalase, and glutathione peroxidase enzymatic activity was increased significantly in the presence of methionine. Bovine mammary epithelial cells also exhibited a certain amount of HSP70 reserve after methionine pretreatment for 24 h, and the expression level of the HSP70 gene and protein further increased with incubation at 42 °C for 30 min. Compared to the control, the expression of HSF-1 mRNA increased, and there was a significantly reduced expression of Bax/Bcl-2 mRNA and a reduced activity of caspase-3 against heat stress. Methionine also increased survival and decreased early apoptosis of hyperthermia-treated BMECs. Thus, methionine has cytoprotective effects on hyperthermia-induced damage in BMECs.     

Mammary glucose uptake in the lactating ewe and the use of methionine arterio-venous difference for the calculation of mammary blood flow

(1) The validity of using the arterio-venous concentration difference of methionine to calculate mammary blood flow in the ewe, on the basis of the Fick principle, is discussed. (2) Calculation of mammary blood flow in the lactating Merino ewe indicated that blood flow per unit weight of tissue and the ratio of blood flow : milk yield were approximately twice that found in the lactating cow and goat. (3) Calculated mammary blood flow in Merino ewes was used in conjunction with glucose arterio-venous difference to determine mammary glucose uptake. Glucose uptake per unit weight of tissue in the ewe was almost double that found in the cow and goat. The ratio of mammary glucose uptake to lactose output was also higher in the ewe than that found in the cow and goat. The utilization of glucose by the mammary gland of the ewe is discussed in relation to the possible greater requirement for NADPH and glycerol for milk fat synthesis in this species.     

MTT比色法检测赖氨酸、蛋氨酸对体外培养的奶牛乳腺上皮细胞增殖的影响

采用噻唑蓝比色法检测赖氨酸、蛋氨酸对体外培养的奶牛乳腺上皮细胞增殖的影响。赖氨酸和蛋氨酸在培养基中的添加浓度分别为0、0.05、0.2、0.4、0.8、1.6、3.2、6.4、12.8、25.6mmol/L和0、0.025、0.1、0.2、0.4、0.8、1.6、3.2、6.4、12.8mmol/L;培养期为24、48和72h。结果表明,赖氨酸在0.8-1.6mmol/L、蛋氨酸在0.4-0.8mmol/L浓度范围内对体外培养的奶牛乳腺上皮细胞增殖的促进作用最明显且在48h时增殖作用最强(P0.0001)。     

Response of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells to tamoxifen, in vitro

Primary cell cultures from a density-defined cell subpopulation of the DMBA-induced rat mammary tumors were exposed to tamoxifen during their log phase of growth. Growth inhibition and the ultrastructure of surviving cells were examined along with the influence of this antiestrogen on the secreted proteins as determined by pulse labeling with [35S]methionine and fluorography. Cell growth was remarkably inhibited at clinically achievable concentrations. However, ultrastructural changes in the surviving cells were minimal, the most noteworthy being the accumulation of myelin bodies. Protein secretion was affected in the defined subpopulation of several tumors by the reduced production of a high-molecular-weight protein. These tumors may represent a population of estrogen-sensitive tumors within the DMBA-induced mammary tumor model.     

Lipotropes alter casein gene expression in bovine mammary acinar culture

Lipotropes (methyl group containing nutrients, including methionine and choline, folic acid, and vitamin B(12)) are essential for cell proliferation and differentiation of mammary tissues. Lipotropes interact in the supply and regulation of intracellular methyl group pools, thereby affecting synthesis and methylation of DNA. To determine the effect of lipotropes on milk protein gene expression, acini isolated from mammary tissues of lactating cows were cultured in one of three treatment media: (1) control, (2) lipotrope deficient, and (3) lipotrope supplemented. beta-Casein mRNA was determined by Northern blotting, and milk protein secretion was measured by a pulse-chase method. The level of beta-casein mRNA was lower in cells grown in lipotrope-deficient medium than in cells grown in the lipotrope-supplemented and control media. Acinar cells cultured in lipotrope-deficient medium also had approximately threefold less milk protein secretion than that of cells in either control or lipotrope-supplemented media. Protein secretion did not differ in the control and lipotrope-supplemented groups. The present study indicates that lipotrope deficiency suppresses total protein secretion and beta-casein gene expression in bovine mammary alveolar epithelial cells in culture.     

Developmental regulation of murine mammary-gland 90 kDa heat-shock proteins.

We have examined the regulation of murine mammary-gland 90 kDa heat-shock protein (hsp-90) as a function of normal development and differentiation. We find that both hsp-90 and amounts of its mRNA are modulated during development and differentiation, with the highest concentrations of mRNA and protein being present in tissues from pregnant and lactating animals respectively. Metabolic labelling experiments with [35S]methionine reveal that the rate of synthesis of hsp-90 also varies among tissues from various developmental states and correlates with the relative hsp-90 mRNA content. These data also suggest that the highest concentration of hsp-90 found in lactating mammary tissues may be due to a greater stability of this protein in this developmental state. The possible significance of the developmental modulation of mammary hsp-90 to mammary steroid-receptor properties is discussed.     

Methionine and serine synergistically suppress hyperhomocysteinemia induced by choline deficiency, but not by guanidinoacetic acid, in rats fed a low casein diet

The effects of dietary supplementation with 0.5% methionine, 2.5% serine, or both on hyperhomocysteinemia induced by deprivation of dietary choline or by dietary addition of 0.5% guanidinoacetic acid (GAA) were investigated in rats fed a 10% casein diet. Hyperhomocysteinemia induced by choline deprivation was not suppressed by methionine alone and was only partially suppressed by serine alone, whereas it was completely suppressed by a combination of methionine and serine, suggesting a synergistic effect of methionine and serine. Fatty liver was also completely prevented by the combination of methionine and serine. Compared with methionine alone, the combination of methionine and serine decreased hepatic S-adenosylhomocysteine and homocysteine concentrations and increased hepatic betaine and serine concentrations and betaine-homocysteine S-methyltransferase activity. GAA-induced hyperhomocysteinemia was partially suppressed by methionine alone, but no interacting effect of methionine and serine was detected. In contrast, GAA-induced fatty liver was completely prevented by the combination of methionine and serine. These results indicate that a combination of methionine and serine is effective in suppressing both hyperhomocysteinemia and fatty liver induced by choline deprivation, and that methionine alone is effective in suppressing GAA-induced hyperhomocysteinemia partially.     

Insulin regulation of amino-acid metabolism in the mammary gland of sheep in early lactation and fed fresh forage

Insulin plays an important role in regulating the partitioning of nutrients to the mammary gland, particularly in lactating ruminants fed concentrate-based diets. There is evidence that the nutritional status of the animals might also affect their response to insulin. This is largely untested in early lactating ruminants fed fresh forage. To investigate nutritional effects on insulin response, 12 lactating sheep, housed indoors, were allocated to one of two treatment groups (hyperinsulinaemic euglycaemic clamp (HEC) or control) in a randomised block design and fed perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) pasture. Mammary amino acid (AA) net uptake from plasma and utilisation for milk protein synthesis was measured during the 4th day of the HEC using arterio-venous concentration differences, and 1-13C-leucine was used to estimate whole body and mammary gland leucine kinetics. There was no change in feed intake, milk protein output and mammary blood flow during the HEC (P > 0.1). The HEC decreased (P < 0.1) the arterial concentrations of all essential AA (EAA) except histidine. The mammary net uptake of some EAA (isoleucine, leucine, methionine and phenylalanine) was reduced by the HEC (P < 0.1). Leucine oxidation in the mammary gland was not altered during the HEC (P > 0.1) but mammary protein synthesis was reduced by the HEC (P < 0.05). These results show that sheep mammary gland can adapt to changing AA precursor supply to maintain milk protein production during early lactation, when fed fresh forage. How this occurs remains unclear, and this area deserves further study.     

Methionine and Serine Synergistically Suppress Hyperhomocysteinemia Induced by Choline Deficiency,but Not by Guanidinoacetic Acid,in Rats Fed a Low Casein Diet

The effects of dietary supplementation with 0.5% methionine, 2.5% serine, or both on hyperhomocysteinemia induced by deprivation of dietary choline or by dietary addition of 0.5% guanidinoacetic acid (GAA) were investigated in rats fed a 10% casein diet. Hyperhomocysteinemia induced by choline deprivation was not suppressed by methionine alone and was only partially suppressed by serine alone, whereas it was completely suppressed by a combination of methionine and serine, suggesting a synergistic effect of methionine and serine. Fatty liver was also completely prevented by the combination of methionine and serine. Compared with methionine alone, the combination of methionine and serine decreased hepatic S-adenosylhomocysteine and homocysteine concentrations and increased hepatic betaine and serine concentrations and betaine-homocysteine S-methyltransferase activity. GAA-induced hyperhomocysteinemia was partially suppressed by methionine alone, but no interacting effect of methionine and serine was detected. In contrast, GAA-induced fatty liver was completely prevented by the combination of methionine and serine. These results indicate that a combination of methionine and serine is effective in suppressing both hyperhomocysteinemia and fatty liver induced by choline deprivation, and that methionine alone is effective in suppressing GAA-induced hyperhomocysteinemia partially.     

Role of Methionine in Cephalosporin Synthesis

Methionine has an almost unique stimulatory effect on biosynthesis of cephalosporins (by Cephalosporium acremonium). No other sulfur-containing compound tested, except dl-methionine-dl-sulfoxide, replaced methionine. dl-Methionine stimulated the synthesis of cephalosporins when added after the growth phase. The utilization of inorganic sulfate was repressed by methionine. Experiments with l-methionine-S(35) showed that essentially all the sulfur in the cephalosporins was derived from methionine. Sulfur-labeled compounds found in the soluble pool from cells grown with methionine-S(35) were methionine, homocysteine, taurine, cystathionine, cysteic acid, glutathionine, and cysteine. dl-Serine-3-C(14) was incorporated into the antibiotics, and its utilization was stimulated by methionine. l-Cysteine had a sparing effect on the incorporation of methionine-S(35) and serine-C(14) into the antibiotics. The data are consistent with the hypothesis that a cystathionine-mediated pathway is operative in the transfer of sulfur between methionine and cysteine.     

Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. I. Radiolabelling of membrane and secretory proteins

A method for the in vitro perfusion of isolated guinea-pig mammary tissue is described that allows the radiolabelling of secretory and membrane proteins. Glands were depleted of methionine, labelled with [35S]methionine for 5 min and perfused with medium containing an excess of unlabelled methionine for varying times. The structural integrity of the alveoli in the perfused glands appeared well maintained. Epithelial polarity was preserved and junctional complexes were evident. About 20% of the methionine provided in the medium was extracted by glands of 10 g wet weight under the labelling conditions employed. With chase periods from 15 to 40 min, 50-70% of the methionine was incorporated into trichloroacetic-acid (TCA)-precipitable material. The principal radiolabelled proteins recovered from the tissue fractions had Mrs and isoelectric points similar to the major secretory proteins (i.e. caseins and alpha-lactalbumin) of guinea-pig milk. Autoradiography of tissue sections at the resolution of the light microscope showed that secretory proteins were transported from sites of synthesis within secretory cells to the alveolar lumina after 45 min. These highly labelled secretory proteins could be almost completely removed from microsomal fractions by treatment with sodium carbonate solutions. Proteins with Mrs from 30 000 to 200 000 were detected in the washed membranes by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. These labelled membrane-associated proteins persisted in the microsomal membrane fraction after chase periods from 7.5 to 40 min.     

Proteins encoded by the long terminal repeat region of mouse mammary tumor virus: identification by hybrid-selected translation.

The long terminal repeat (LTR) region of mouse mammary tumor virus (MMTV) is known to contain an open reading frame of sufficient length to code for a protein of 36,000 Mr. The coding capacity of the 3' sequences of MMTV genomic RNA has been demonstrated by in vitro translation studies, which have reported the synthesis of four related proteins: p36, p24, p21, and p18. These proteins are overlapping translation products of the same open reading frame, with the smaller ones initiating at internal methionine codons. From the predicted amino acid sequence of the LTR protein, we have selected a region likely to be antigenic, obtained a synthetic peptide of that region, and raised antiserum to the peptide. The antipeptide serum specifically immunoprecipitated all four proteins from in vitro translated genomic 3' MMTV RNA, plus an additional one of 32,000 Mr. Published sequence data of MMRV LTRs show an internal AUG codon at a position which could initiate a protein of 32,000 Mr. The three smaller in vitro translation products (p24, p21, and p18) were consistently synthesized in much greater amounts than the p36 or p32 protein. The relative amount of each in vitro synthesized protein from genomic MMTV RNA could be predicted and was in good agreement with the postulated effect of flanking nucleotides on the efficiency of the respective AUG initiation codon. Polyadenylated RNAs, isolated from various mouse tissues, were selected by hybridization to plasmid DNA containing MMTV LTR sequences immobilized on nitrocellulose. In vitro translation of hybrid-selected mRNAs isolated from BALB/c mouse lactating mammary glands and carcinogen-induced mammary tumors, followed by immunoprecipitation with antipeptide serum, revealed that only one polypeptide was synthesized by the MMTV LTR-specific mRNA, the 36,000 Mr species.     

The spatial structure of the axially bound methionine in solution conformations of horse ferrocytochrome c and Pseudomonas aeruginosa ferrocytochrome c 551 by 1H NMR

A generally applicable method for the determination of the spatial structure of the heme iron-bound methionine in c-type ferrocytochromes at atomic resolution is presented. It relies primarily on measurements of nuclear Overhauser effects between the individual hydrogen atoms of the axial methionine, and between individual hydrogens of the methionine and the heme group. Four different methionine conformers, corresponding to the four possible stereospecific assignments for the methionine methylene proton resonances, are generated by a structural interpretation of the nuclear Overhauser effects with the use of an interactive computer graphics technique. A unique structure and unique stereospecific resonance assignments are then obtained by discriminating between these four conformers on the basis of van der Waals' constraints and heme ring current effects on the chemical shifts. The use of the method is illustrated with studies of horse ferrocytochrome c and Pseudomonas aeruginosa ferrocytochrome c 551. Comparison with the crystal structures shows close coincidence between the methionine conformations in solution and in single crystals of these proteins.Abbreviations NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - TOE truncated driven nuclear Overhauser effect     

Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. II. Biogenesis of milk-fat-globule membrane proteins

Guinea-pig mammary tissue was perfused in vitro, radiolabelled with [35S]methionine and intracellular protein precursors of the milk-fat-globule membrane (FGM) recovered by immunoabsorption techniques. Labelled xanthine oxidase was solely detected in post-microsomal supernatants and butyrophilin in carbonate-washed membranes. A major glycoprotein (Gp 55), was initially present in a membrane-bound form, but after longer perfusion times a fraction of this protein was recovered in the post-microsomal supernatant. These results are discussed with reference to formation of the apically-derived FGM.     

A formylated hexapeptide ligand mimics the ability of Wnt-5a to impair migration of human breast epithelial cells

Loss of Wnt-5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt-5a, we identified 14 peptide fragments and investigated their ability to mimic the effects of Wnt-5a on mammary cell adhesion and migration. Two of these peptides significantly increased adhesion and impaired migration in the non-tumorigenic HB2 breast epithelial cell line and in the MDA-MB-468 breast cancer cell line, both of which show little endogenous expression of the Wnt-5a protein. We removed two amino acids at a time from the N terminus of the shorter of these two peptides to identify the shortest peptide that still inhibited migration. The influence on tumor cell adhesion was gradually lost and was no longer detectable when only six amino acids remained. However, formylation of the N-terminal methionine of this hexapeptide restored its effect on adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism, even at a low pH such as encountered in breast tumor tissue. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, whereas it did not affect the cellular levels of unphosphorylated beta-catenin or active JNK. The novel formyl-Met-Asp-Gly-Cys-Glu-Leu peptide ligand is not only a valuable experimental tool but has also a potential role in antimetastatic treatment of the 50% of human breast cancer patients that have reduced endogenous Wnt-5a protein expression.     

Inhibition of Growth of Escherichia coli and of Homoserine O-Transsuccinylase by α-Methylmethionine

The methionine analogue, alpha-methylmethionine, inhibits bacterial growth, but its action is overcome by methionine, homocysteine, and cystathionine. The effect of the analogue on growth is attributed to its ability to mimic methionine as a feed-back inhibitor of the first enzyme specific to methionine biosynthesis. This conclusion is based on the findings that (i) alpha-methylmethionine inhibits excretion of O-succinylhomoserine, the product of the first enzyme, by a methionine auxotroph unable to convert succinylhomoserine to cystahionine, and that (ii) the enzyme homoserine O-transsuccinylase is inhibited by alpha-methylmethionine in extracts of Escherichia coli. alpha-Methylmethionine also inhibits methionyl-ribonucleic acid synthetase in extracts, but this inhibition probably does not affect growth.     

Selenium retention and inhibition of cell growth in mouse mammary epithelial cell lines in vitro

The steady state levels of growth inhibitory doses of inorganic selenium were examined in five different mammary epithelial cell lines: MOD, COMMA-D, COMMA-F, COMMA-T, and YN-4. The retention of selenium was monitored using a radioactive isotope,⁷⁵Se. Growth inhibition correlated with high levels of selenium in the cell. Generally, the retention of intracellular selenium was not dependent upon cell density, cell number, net growth rate, or tumorigenicity of the mammary cell lines. One cell line, COMMA-D, exhibited an unique response wherein the amount of selenium retained was low and the growth inhibitory effects of selenium were negligible when the cells were exposed to selenium at low density. However, at high cell densities, the COMMA-D cells responded like the other four cell lines. The growth inhibitory effect of selenium was reversible; upon removal of selenium from the medium, cells start synthesizing DNA within 24h. The retention of selenium was influenced by constituents in the growth medium. In particular, cysteine, but not methionine, purines, or pyrimidines altered selenium retention and counteracted the growth inhibitory effects of selenium. These results indicated that the mammary cell lines, particulary COMMA-D and MOD are good model systems to examine the uptake, retention, localization, and function of inorganic selenium under conditions where it acts as a growth inhibitory agent.