Biomedical subjects

Margaret C Neville

Publications and source records attributed to Margaret C Neville.

16 recordsLinked to original sources

PubMed2006

Metabolic regulation in the lactating mammary gland: a lipid synthesizing machine.

The mammary gland of the lactating mouse synthesizes and secretes milk lipid equivalent to its entire body weight in a single 20-day lactation cycle, making it one of the most active lipid synthetic organs known. We test the hypothesis that multiple control points and potential regulatory mechanisms regulate milk lipid synthesis at the level of gene expression. The mammary transcriptome of 130 genes involved in glucose metabolism was examined at late pregnancy and early lactation, utilizing data obtained from microarray analysis of mammary glands from quadruplicate FVB mice at pregnancy day 17 and lactation day 2. To correlate changes with physiological parameters, the metabolome obtained from magnetic resonance spectroscopy of flash-frozen glands at day 17 of pregnancy was compared with that at day 2 of lactation. A significant increase in carbohydrates (glucose, lactose, sialic acid) and amino acids (alanine, aspartate, arginine, glutamate) with a moderate increase in important osmolytes (myo-inositol, betaine, choline derivatives) were observed in the lactating gland. In addition, diets containing 8% or 40% lipid were fed from lactation days 5-10 and mammary glands and livers of triplicate FVB mice prepared for microarray analysis. The results show that substantial regulation of lipid synthesis occurs at the level of mRNA expression and that some of the regulation points differ substantially from the liver. They also implicate the transcription factor SREBP-1c in regulation of part of the pathway.

Animals↗

PubMed2006

Analysis of lactation defects in transgenic mice.

Although lactation is the only physiological function of the mammary gland, little is known about the molecular events required for secretory activation and milk production. Genetically altered mice have been used extensively to study mammary gland development during puberty and pregnancy, as well as mammary tumorigenesis. A number of approaches have been used to produce genetic modifications in mammary glands of mice, including transgenic mice utilizing mammary specific promoters, traditional knockout mice, mammary-specific gene deletion, and conditionally-regulated transgenes. The same technologies can be used to study secretory activation and lactation; however only a comparatively small number of studies to date have used these approaches to study these events. In this paper we review the technologies available to make genetically modified mice for the study of secretory activation and lactation as well as specific analytical procedures that can be used to characterize mice with lactation defects.

Animals↗

PubMed2005

Claudin 7 expression and localization in the normal murine mammary gland and murine mammary tumors.

INTRODUCTION: Claudins, membrane-associated tetraspanin proteins, are normally associated with the tight junctions of epithelial cells where they confer a variety of permeability properties to the transepithelial barrier. One member of this family, claudin 7, has been shown to be expressed in the human mammary epithelium and some breast tumors. To set the stage for functional experiments on this molecule, we examined the developmental expression and localization of claudin 7 in the murine mammary epithelium and in a selection of murine mammary tumors. METHOD: We used real-time polymerase chain reaction, in situ mRNA localization, and immunohistochemistry (IHC) to examine the expression and localization of claudin 7. Frozen sections were examined by digital confocal microscopy for colocalization with the tight-junction protein ZO1. RESULTS: Claudin 7 was expressed constitutively in the mammary epithelium at all developmental stages, and the ratio of its mRNA to that of keratin 19 was nearly constant through development. By IHC, claudin 7 was located in the basolateral part of the cell where it seemed to be localized to discrete vesicles. Scant colocalization with the tight-junction scaffolding protein ZO1 was observed. Similar results were obtained from IHC of the airway epithelium and some renal tubules; however, claudin 7 did partly colocalize with ZO1 in EPH4 cells, a normal murine mammary cell line, and in the epididymis. The molecule was localized in the cytoplasm of MMTV-neu and the transplantable murine tumor cell lines TM4, TM10, and TM40A, in which its ratio to cytokeratin was higher than in the normal mammary epithelium. CONCLUSION: Claudin 7 is expressed constitutively in the mammary epithelium at approximately equal levels throughout development as well as in the murine tumors examined. Although it is capable of localizing to tight junctions, in the epithelia of mammary gland, airway, and kidney it is mostly or entirely confined to punctate cytoplasmic structures, often near the basolateral surfaces of the cells and possibly associated with basolateral membranes. These observations suggest that claudin 7 might be involved in vesicle trafficking to the basolateral membrane, possibly stabilizing cytoplasmic vesicles or participating in cell-matrix interactions.

Animals↗

PubMed2005

Calcium secretion into milk.

Ionized calcium ([Ca(2+)]) is present in milk at concentrations around 3 mM, a concentration that drives the formation of complexes with citrate, phosphate, and casein, thereby generating compounds that carry the major portion of calcium in milk. In humans and cows, where it has been studied, changes in milk calcium appear to be regulated by the amount of citrate and casein in milk rather than changes in [Ca(2+)]. Most or all of the calcium in milk is likely derived through exocytosis of secretory vesicles derived from the Golgi compartment where a calcium ATPase mediates transport from the cytoplasm. The identity of the transporters is not yet certain but gene expression for the plasma membrane calcium ATPase, PMCA2bw, and the secretory pathway calcium ATPase, SPCA, is highly upregulated during lactation. Currently nothing appears to be known about the mechanisms that mediate transport of calcium across the basolateral membrane of the alveolar cell.

Animals↗

PubMed2005

Breast milk retinol concentrations are not associated with systemic inflammation among breast-feeding women in Malawi.

The acute phase response and inflammation are associated with lower plasma retinol concentrations, but their effect on breast milk retinol concentrations is unclear. We measured plasma retinol concentrations, acute phase proteins, and breast milk retinol concentrations in 237 breast-feeding women at 2 wk postpartum in Blantyre, Malawi; 16.5% of the women had plasma retinol < 0.70 micromol/L and 14.8% had breast milk retinol < 1.05 micromol/L. Among women with and without inflammation [alpha(1)-acid glycoprotein (AGP) > 1 g/L and/or C-reactive protein (CRP) > 5 mg/L], geometric mean (95% CI) plasma retinol was 0.89 (0.84, 0.94) and 1.05 (1.01, 1.17) mumol/L, respectively (P < 0.0001). Among women with and without inflammation, geometric mean (95% CI) breast milk retinol was 2.12 (1.89, 2.36) and 2.05 (1.75, 2.39) micromol/L, respectively (P = 0.74). In multiple linear regression models adjusting for age, parity, education, BMI, and days postpartum, plasma retinol concentrations were associated with plasma AGP and CRP concentrations (P < 0.0001 and P = 0.01, respectively), whereas breast milk retinol concentrations were unaffected by plasma AGP and CRP concentrations (P = 0.22 and P = 0.86, respectively). These findings suggest that breast milk retinol concentrations are not affected by systemic inflammation.

Adult↗

PubMed2005

Status of carotenoids, vitamin A, and vitamin E in the mother-infant dyad and anthropometric status of infants in Malawi.

This prospective study was carried out during February 2000-April 2003 to characterize the relationship between the status of carotenoids, vitamin E, and retinol and anthropometric status in apparently healthy infants and their mothers in Blantyre, Malawi. Anthropometric status of infants and concentrations of carotenoids (alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, zeaxanthin, and lycopene), retinol, and alpha-tocopherol in plasma were measured in 173 infants at 12 months of age, and concentrations of carotenoids, retinol, and a-tocopherol in plasma were measured in their mothers two weeks postpartum. In multivariate analyses, concentrations of retinol, total carotenoids, non-provitamin A carotenoids, and alpha-tocopherol in infants were associated with under-weight (p = 0.05). Concentrations of a-tocopherol were associated with wasting (p = 0.04). Concentrations in mothers and infants were all correlated (correlation coefficients from 0.230 to 0.502, p < 0.003). The findings suggest that poor status of carotenoids, retinol, and alpha-tocopherol in infants is associated with their poor anthropometric status, and status of carotenoids, retinol, and alpha-tocopherol in mothers and infants has a low-to-moderate association in the mother-infant dyad.

Adult↗

PubMed2004

Bovine glucose transporter GLUT8: cloning, expression, and developmental regulation in mammary gland.

GLUT8 is a newly identified member of the facilitative glucose transporter family, which characteristically exhibits high-affinity glucose transport activity. The expression of GLUT8 has been shown to depend on gonadotropin secretion in human testes and to be regulated by insulin in the blastocyst. To characterize GLUT8 and investigate its role in normal mammary gland function, we cloned and sequenced the full-length cDNA of bovine GLUT8. The 2073-base-pair cDNA sequence is predicted to encode a protein of 478 amino acids, with a molecular weight of approximately 51 kDa. The deduced amino acid sequence of bovine GLUT8 is 90%, 84%, 84% and 58% identical to human, mouse, rat and chicken GLUT8, and is 26%, 27% and 24% identical to bovine GLUT1, GLUT3 and GLUT4, respectively. Bovine GLUT8 retains the characteristic structural features of GLUT8 proteins previously identified from other species including membrane spanning helices, glucose transporter motifs, an N-linked glycosylation site on loop 9 and a putative dileucine internalization motif. The major in vitro transcription and translation product of bovine GLUT8 cDNA migrated at an apparent molecular weight of 38 kDa similar to the sizes reported for GLUT8 from other mammalian species. In the presence of canine microsomal membranes, the translation product increased to 40 kDa suggesting glycosylation. Transient transfection studies using a FLAG epitope tagged construct in COS-7 cells revealed that bovine GLUT8 is localized to the cytoplasm in non-stimulated conditions. A 2.1-kb GLUT8 mRNA transcript was detected at high levels in bovine testes, at moderate levels in lactating bovine mammary gland, lung, kidney, spleen, intestine and skeletal muscle, and at low levels in bovine liver. GLUT8 mRNA expression in bovine mammary gland increased about 10-fold (P<0.001) during late pregnancy and early lactation, similar to the pattern of change in GLUT1 mRNA and more dramatic than the increase seen in mouse mammary gland. These results suggest that GLUT8 expression may be regulated by lactogenic hormones and that GLUT8 may play a role in glucose uptake in the lactating mammary gland.

Amino Acid Sequence↗

PubMed2004

Albumin transcytosis across the epithelium of the lactating mouse mammary gland.

Murine milk contains 18 mg ml(-1) serum albumin, a concentration equal to that in the serum of the lactating mouse. We examined cellular transport using in vivo methods in the mouse. At steady state the specific activity of (125)I-albumin injected into the blood stream was equal in plasma and whey, confirming that milk albumin is extra-mammary in origin. Fluorescent albumin crossed the gland from basolateral surface to lumen via cytoplasmic vesicles, but was not transported in the apical to basal direction. Albumin was segregated from transferrin at the basal surface of the epithelial cells and did not colocalize with either caveolin-1 or -2. Vesicular transport was not disrupted by filipin providing additional evidence that, unlike the vascular endothelium, caveoli are not involved. Cytoplasmic albumin was localized to vesicles containing IgA and transport was disrupted by agents that interfere with clathrin-mediated endocytosis. Together, these findings provide evidence that albumin is transported across the mammary epithelium by the same pathway as immunoglobulin. The possibility that the massive transfer of albumin into mouse milk is mediated by fluid phase transport is considered.

Animals↗

PubMed2004

Regulation of milk lipid formation and secretion in the mouse mammary gland.

Cytosolic lipid droplets (CLDs), the immediate precursors of milk lipids in lactating animals, undergo cell-specific changes in their formation and intracellular distribution during mammary gland differentiation. Cell biological studies indicate that CLD formation in mammary epithelial cells is regulated in part by AKT-dependent increases in glucose uptake. Proteomic studies show that CLDs from lactating mammary epithelial cells possess a distinct protein composition enriched in molecules involved in their secretion and intracellular transport. CLD secretion is dependent on lactation and requires the purine catabolic enzyme xanthine oxidoreductase (XOR). Confocal immunofluorescence microscopy of XOR in lactating and nonlactating mammary glands and biochemical analysis of secreted CLDs link the secretion process to the formation of a stable tripartite complex between XOR, adipophilin (ADPH), and butyrophilin (Btn). Together these studies provide a molecular and cellular framework for understanding the process of milk lipid formation.

Animals↗

PubMed2003

Mammary physiology and milk secretion.

The presence of drugs or other potentially toxic materials in milk is an obvious public health risk, especially to infants and neonates. There is also increasing concern that human breast cancer is principally epigenetic in origin and results from environmentally produced lesions. Little is known about the mechanisms by which toxic substances enter milk or mammary tissue but knowledge of these processes is important to toxicologists and researchers involved in drug design and metabolism. Five general pathways have been described for transport of proteins, lipids, ions, nutrients and water into milk. Four of these pathways are transcellular, involving transport across at least two membrane barriers; the fifth is paracellular and allows direct exchange of interstitial and milk components. Solute transport by these pathways is mediated by a diverse, and complex array of transport and secretory processes that are regulated by hormonal, developmental, and physiological factors. Current research is beginning to define the mechanisms underlying some of these processes, however the regulation and coordination of solute transport mechanisms remains poorly understood. In this article we review our current understanding of the normal solute transport and secretory processes involved in milk production, and discuss potential regulatory mechanisms.

Animals↗

PubMed2003

Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation.

Expression of constitutively activated Akt in the mammary glands of transgenic mice results in a delay in post-lactational involution. We now report precocious lipid accumulation in the alveolar epithelium of mouse mammary tumor virus-myr-Akt transgenic mice accompanied by a lactation defect that results in a 50% decrease in litter weight over the first 9 days of lactation. Although ductal structures and alveolar units develop normally during pregnancy, cytoplasmic lipid droplets appeared precociously in mammary epithelial cells in early pregnancy and were accompanied by increased expression of adipophilin, which is associated with lipid droplets. By late pregnancy the lipid droplets had become significantly larger than in nontransgenic mice, and they persisted into lactation. The fat content of milk from lactating myr-Akt transgenic mice was 65-70% by volume compared to 25-30% in wild-type mice. The diminished growth of pups nursed by transgenic mothers could result from the high viscosity of the milk and the inability of the pups to remove sufficient quantities of milk by suckling. Transduction of the CIT3 mammary epithelial cell line with a recombinant human adenovirus encoding myr-Akt resulted in an increase in glucose transport and lipid biosynthesis, suggesting that Akt plays an important role in regulation of lipid metabolism.

Animals↗

PubMed2003

Functional development of the mammary gland: use of expression profiling and trajectory clustering to reveal changes in gene expression during pregnancy, lactation, and involution.

To characterize the molecular mechanisms by which progesterone withdrawal initiates milk secretion, we examined global gene expression during pregnancy and lactation in mice, focusing on the period around parturition. Trajectory clustering was used to profile the expression of 1358 genes that changed significantly between pregnancy day 12 and lactation day 9. Predominantly downward trajectories included stromal and proteasomal genes and genes for the enzymes of fatty acid degradation. Milk protein gene expression increased throughout pregnancy, whereas the expression of genes for lipid synthesis increased sharply at the onset of lactation. Examination of regulatory genes with profiles similar or complementary to those of lipid synthesis genes led to a model in which progesterone stimulates synthesis of TGF-beta, Wnt 5b, and IGFBP-5 during pregnancy. These factors are suggested to repress secretion by interfering with PRL and IGF-1 signaling. With progesterone withdrawal, PRL and IGF-1 signaling are activated, in turn activating Akt/PKB and the SREBPs, leading to increased lipid synthesis.

Animals↗

PubMed2003

Transduction of the mammary epithelium with adenovirus vectors in vivo.

Because the mammary parenchyma is accessible from the exterior of an animal through the mammary duct, adenovirus transduction holds promise for the short-term delivery of genes to the mammary epithelium for both research and therapeutic purposes. To optimize the procedure and evaluate its efficacy, an adenovirus vector (human adenovirus type 5) encoding a green fluorescent protein (GFP) reporter and deleted of E1 and E3 was injected intraductally into the mouse mammary gland. We evaluated induction of inflammation (by intraductal injection of [(14)C]sucrose and histological examination), efficiency of transduction, and maintenance of normal function in transduced cells. We found that transduction of the total epithelium in the proximal portion of the third mammary gland varied from 7% to 25% at a dose of 2 x 10(6) PFU of adenovirus injected into day 17 pregnant mice. Transduction was maintained for at least 7 days with minimal inflammatory response; however, significant mastitis was observed 12 days after transduction. Adenovirus transduction could also be used in the virgin animal with little mastitis 3 days after transduction. Transduced mammary epithelial cells maintained normal morphology and function. Our results demonstrate that intraductal injection of adenovirus vectors provides a versatile and noninvasive method of investigating genes of interest in mouse mammary epithelial cells.

Adenoviridae Infections↗

PubMed2002

Nuclear import of PKCdelta is required for apoptosis: identification of a novel nuclear import sequence.

We have shown previously that protein kinase Cdelta (PKCdelta) is required for mitochondrial-dependent apoptosis. Here we show that PKCdelta is imported into the nucleus of etoposide-treated cells, that nuclear import is required for apoptosis and that it is mediated by a nuclear localization signal (NLS) in the C-terminus of PKCdelta. Mutation of the caspase cleavage site of PKCdelta inhibits nuclear accumulation in apoptotic cells, indicating that caspase cleavage facilitates this process. Expression of the PKCdelta catalytic fragment (CFdelta) in transfected cells results in nuclear localization and apoptosis. We show that the PKCdelta NLS is required for nuclear import of both full-length PKCdelta and CFdelta, and drives nuclear localization of a multimeric green fluorescent protein. Mutations within the NLS of CFdelta prevent nuclear accumulation and block apoptosis. Conversely, nuclear expression of a kinase-negative catalytic fragment (KN-CFdelta) protects cells from etoposide-induced apoptosis. Mutation of the NLS blocks the ability of KN-CFdelta to protect against etoposide-induced apoptosis. These results indicate that PKCdelta regulates an essential nuclear event(s) that is required for initiation of the apoptotic pathway.

Active Transport, Cell Nucleus↗

PubMed2002

Sodium dependence of nitrofurantoin active transport across mammary epithelia and effects of dipyridamole, nucleosides, and nucleobases.

PURPOSE: The sodium dependence and effects of nucleoside and nucleobase transport inhibitors were determined to ascertain the role of sodium dependent nucleoside or nucleobase transporters in nitrofurantoin active transport across mammary epithelia. METHODS: Five lactating female rats received steady-state intravenous infusions of nitrofurantoin with and without the broad-based inhibitor dipyridamole. In the CIT3 murine model of lactation, 14C-nitrofurantoin basolateral to apical permeability was examined in the presence of varying sodium concentrations, purine and pyrimidine nucleosides and nucleobases, and dipyridamole. RESULTS: Dipyridamole effectively inhibited 14C-nitrofurantoin flux across CIT3 cells, with Ki = 0.78 microM (95% C.I. = 0.11 to 5.3 microM) and significantly decreased the milk-to-serum ratio of nitrofurantoin from 29.2 +/- 5.0 to 11.0 +/- 6.3 without changing systemic clearance. Nitrofurantoin active transport was significantly inhibited by complete sodium replacement. Adenosine and guanosine significantly inhibited nitrofurantoin permeability (54.5 +/- 2.6 (microl/hr)/cm2 and 50.7 +/- 0.6 (microl/hr)/cm2, respectively, vs. control 90.5 +/- 4.6 (microl/hr)/cm2) but uridine, thymidine, and the nucleobases had no effect. CONCLUSIONS: Nitrofurantoin active transport was sodium dependent and inhibited by dipyridamole, adenosine, and guanosine, but known sodium dependent nucleoside or nucleobase transporters were not involved.

Animals↗

PubMed2002

Hormonal regulation of mammary differentiation and milk secretion.

The endocrine system coordinates development of the mammary gland with reproductive development and the demand of the offspring for milk. Three categories of hormones are involved. The levels of the reproductive hormones, estrogen, progesterone, placental lactogen, prolactin, and oxytocin, change during reproductive development or function and act directly on the mammary gland to bring about developmental changes or coordinate milk delivery to the offspring. Metabolic hormones, whose main role is to regulate metabolic responses to nutrient intake or stress, often have direct effects on the mammary gland as well. The important hormones in this regard are growth hormone, corticosteroids, thyroid hormone, and insulin. A third category of hormones has recently been recognized, mammary hormones. It currently includes growth hormone, prolactin, PTHrP, and leptin. Because a full-term pregnancy in early life is associated with a reduction in breast carcinogenesis, an understanding of the mechanisms by which these hormones bring about secretory differentiation may offer clues to the prevention of breast cancer.

Animals↗