Differential expression and localization of lipid transporters in the bovine mammary gland during the pregnancy-lactation cycle

The transport of lipids across mammary gland epithelial cells (MEC) determines milk lipid content and composition. We investigated the expression of lipid transporters and their regulators in comparison to blood metabolites during lactation and dry period (DP) in dairy cows. Repeated mammary gland biopsies and blood samples were taken from 10 animals at 7 stages of the pregnancy-lactation cycle. Expression levels of the specific mRNAs were determined by quantitative reverse transcription-PCR, whereas ABCA1 was localized by immunohistochemistry. Blood serum metabolites were determined by common enzymatic chemistries. Elevated mRNA profiles of ABCA1 and ABCA7 were found during DP as compared with lactation and were inversely associated with blood cholesterol levels. Elevated levels of ABCG2, NPC1, SREBP1, SREBP2, LXR alpha, and PPAR gamma were found postpartum, whereas ABCG1 did not differ between the functional stages of the mammary gland. The ABCA1 protein was localized in MEC and showed differential activity between DP and lactation suggesting a role of ABCA1 in the removal of excess cellular cholesterol from MEC during the DP. The expression profiles of ABCA7 and NPC1 may reflect a role of these transporters in the clearance of apoptotic cells and the intracellular redistribution of cholesterol, respectively. Regulation of lipid transporters in the mammary gland is partially associated with transcription factors that control lipid homeostasis.

Deregulated ephrin-B2 expression in the mammary gland interferes with the development of both the glandular epithelium and vasculature and promotes metastasis formation

Eph receptor tyrosine kinases and their membrane-bound ephrin ligands play key roles during morphogenesis and adult tissue homeostasis. Receptor-ligand interactions result in forward and reverse signalling from the receptor and ligand respectively. To delineate the role(s) of forward and reverse signalling in mammary gland biology we have established transgenic mice exhibiting mammary epithelial-specific overexpression of either the native ephrin-B2 or a dominant negative ephrin-B2 mutant incapable of reverse signalling. During pregnancy and lactation overexpression of the native ephrin-B2 resulted in precocious differentiation, whereas overexpression of mutated ephrin-B2 caused delayed epithelial differentiation and in disturbed tissue architecture. Both transgenes affected also mammary vascularisation. Whereas ephrin-B2 induced superfluous but organised capillaries, mutant ephrin-B2 overexpression resulted in an irregular vasculature with blind-ending capillaries. Mammary tumours were not observed in either transgenic line, however, the crossing with NeuT transgenic animals revealed that mutated ephrin-B2 expression significantly accelerated tumour growth and imposed a metastatic phenotype.

Insect bite hypersensitivity in the horse: comparison of IgE-binding proteins in salivary gland extracts from Simulium vittatum and Culicoides nubeculosus

Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis of horses caused by bites of insects such as Culicoides or Simulium spp. The aim of the present study was to compare the IgE-binding pattern of sera of IBH-affected horses to Culicoides nubeculosus and Simulium vittatum salivary gland extracts (SGE). Individual IgE responses to proteins of S. vittatum and C. nubeculosus SGEs were evaluated in 15 IBH-affected and three healthy horses on immunoblots. Fourteen out of the 15 IBH-affected but none of the healthy horses showed individual IgE binding patterns to seven and six main protein bands in C. nubeculosus and S. vittatum SGE, respectively. These 14 sera showed IgE-binding to proteins from SGE of both C. nubeculosus and S. vittatum, but they reacted with fewer protein bands derived from S. vittatum than from C. nubeculosus SGE. Sera showing IgE-binding to a 32 kDa band from C. nubeculosus always bound to a 32 kDa band from S. vittatum. Similarly, all sera binding to a 70 kDa band from C. nubeculosus reacted with a corresponding band in S. vittatum SGE. The 70 kDa bands from S. vittatum and C. nubeculosus were identified by mass spectrometry as heat shock protein-70-cognate-3.

Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

Cholesterol Transport and Regulation in the Mammary Gland.

The milk-producing alveolar epithelial cells secrete milk that remains after birth the principal source of nutrients for neonates. Milk secretion and composition are highly regulated processes via integrated actions of hormones and local factors which involve specific receptors and downstream signal transduction pathways. Overall milk composition is similar among mammalian species, although the content of individual constituents such as lipids may significantly differ from one species to another. The milk lipid fraction is essentially composed of triglycerides, which represent more than 95 % of the total lipids in human and commercialized bovine milk. Though sterols, including cholesterol, which is the major milk sterol, represent less than 0.5 % of the total milk lipid fraction, they are of key importance for several biological processes. Cholesterol is required for the formation of biological membranes especially in rapidly growing organisms, and for the synthesis of sterol-based compounds. Cholesterol found in milk originates predominantly from blood uptake and, to a certain extent, from local synthesis in the mammary tissue. The present review summarizes current knowledge on cellular mechanisms and regulatory processes determining intra- and transcellular cholesterol transport in the mammary gland. Cholesterol exchanges between the blood, the mammary alveolar cells and the milk, and the likely role of active cholesterol transporters in these processes are discussed. In this context, the hormonal regulation and signal transduction pathways promoting active cholesterol transport as well as potential regulatory crosstalks are highlighted.

Nutrient Transport in the Mammary Gland: Calcium, Trace Minerals and Water Soluble Vitamins.

Milk nutrients are secreted by epithelial cells in the alveoli of the mammary gland by several complex and highly coordinated systems. Many of these nutrients are transported from the blood to the milk via transcellular pathways that involve the concerted activity of transport proteins on the apical and basolateral membranes of mammary epithelial cells. In this review, we focus on transport mechanisms that contribute to the secretion of calcium, trace minerals and water soluble vitamins into milk with particular focus on the role of transporters of the SLC series as well as calcium transport proteins (ion channels and pumps). Numerous members of the SLC family are involved in the regulation of essential nutrients in the milk, such as the divalent metal transporter-1 (SLC11A2), ferroportin-1 (SLC40A1) and the copper transporter CTR1 (SLC31A1). A deeper understanding of the physiology and pathophysiology of these transporters will be of great value for drug discovery and treatment of breast diseases.

The Great Silk Alternative Multiple Co-Evolution of Web Loss and Sticky Hairs in Spiders

Spiders are the most important terrestrial predators among arthropods. Their ecological success is reflected by a high biodiversity and the conquest of nearly every terrestrial habitat. Spiders are closely associated with silk, a material, often seen to be responsible for their great ecological success and gaining high attention in life sciences. However, it is often overlooked that more than half of all Recent spider species have abandoned web building or never developed such an adaptation. These species must have found other, more economic solutions for prey capture and retention, compensating the higher energy costs of increased locomotion activity. Here we show that hairy adhesive pads (scopulae) are closely associated with the convergent evolution of a vagrant life style, resulting in highly diversified lineages of at least, equal importance as the derived web building taxa. Previous studies often highlighted the idea that scopulae have the primary function of assisting locomotion, neglecting the fact that only the distal most pads (claw tufts) are suitable for those purposes. The former observations, that scopulae are used in prey capture, are largely overlooked. Our results suggest the scopulae evolved as a substitute for silk in controlling prey and that the claw tufts are, in most cases, a secondary development. Evolutionary trends towards specialized claw tufts and their composition from a low number of enlarged setae to a dense array of slender ones, as well as the secondary loss of those pads are discussed further. Hypotheses about the origin of the adhesive setae and their diversification throughout evolution are provided.

Spider Ecophysiology

With over 43,000 species, spiders are the largest predacious arthropod group. They have developed key characteristics such as multi-purpose silk types, venoms consisting of hundreds of components, locomotion driven by muscles and hydraulic pressure, a highly evolved key-lock mechanism between the complex genital structures, and many more unique features. After 300 million years of evolutionary refinement, spiders are present in all land habitats and represent one of the most successful groups of terrestrial organisms. Ecophysiology combines functional and evolutionary aspects of morphology, physiology, biochemistry and molecular biology with ecology. Cutting-edge science in spiders focuses on the circulatory and respiratory system, locomotion and dispersal abilities, the immune system, endosymbionts and pathogens, chemical communication, gland secretions, venom components, silk structure, structure and perception of colours as well as nutritional requirements. Spiders are valuable indicator species in agroecosystems and for conservation biology. Modern transfer and application technologies research spiders and their products with respect to their value for biomimetics, material sciences, and the agrochemical and pharmaceutical industries.

Myoepithelioma of the cerebellopontine angle: a previously not documented benign salivary gland-type neoplasm within the cranium

Myoepithelioma is a dimorphic neoplasm with contractile-epithelial phenotype, originally interpreted as deriving from, but not actually restricted to the salivary glands. As a novel addition to the list of exquisitely rare intracranial salivary gland-type tumors and tumor-like lesions, we report on an example of myoepithelioma encountered in the left cerebellopontine angle of a 32-year-old male. Clinically presenting with ataxia and dizziness, this extraaxial mass of 4 × 3.5 × 3 cm was surgically resected, and the patient is alive 6 years postoperatively. Histologically, the tumor exhibited a continuum ranging from compact fascicles of spindle cells to epithelial nests and trabeculae partitioned by hyalinized septa, while lacking tubular differentiation. Regardless of architectural variations, there was robust immunoexpression of S100 protein, smooth muscle actin, GFAP, cytokeratin, and vimentin. Cytologic atypia tended to be modest throughout, and the MIB1 labeling index averaged less than 1%. Fluorescent in situ hybridization indicated no rearrangement of the EWSR1 locus. We interpret these results to suggest that myoepithelioma of the posterior fossa - along with related salivary epithelial tumors in this ostensibly incongruous locale - may possibly represent analogous neoplasms to their orthotopic counterparts, ones arising within aberrant salivary anlagen. The presence of the latter lends itself to being mechanistically accounted for by either postulating placodal remnants in the wake of branchial arch development, or linking them to exocrine glandular nests within endodermal cysts. Alternatively, myoepithelioma at this site could be regarded as a non tissue-specific lesion similar to its relatives ubiquitously occurring in the soft parts.

Book Symposium The Electronic Silk Road: Connecting Dots in a World of Bits

This is a contribution to a book symposium on The Electronic Silk Road by Anupam Chander (Yale University Press).

The mammary gland in small ruminants: major morphological and functional events underlying milk production – a review

The importance of small ruminants to the dairy industry has increased in recent years, especially in developing countries, where it has a high economic and social impact. Interestingly and despite the fact that the mammary gland is the specialised milk production organ, very few authors studied the modifications occurring in the mammary gland through the lactation period in production animals, particularly in the small ruminants, sheep (Ovis aries) and goat (Capra hircus). Nevertheless, understanding the different mammary gland patterns throughout lactation is essential to improve dairy production. In addition, associating these patterns with different milking frequencies, lactation number or different diets is also of high importance, directly affecting the dairy industry. The mammary gland is commonly composed of parenchyma and stroma, which includes the ductal system, with individual proportions of each changing during the different periods and yields in a lactation cycle. Indeed, during late gestation, as well as during early to mid-lactation, mammary gland expansion occurs, with an increase in the number of epithelial cells and lumen area, which leads to increment of the parenchyma tissue, as well as a reduction of stroma, corresponding macroscopically to the increase in mammary gland volume. Throughout late lactation, the mammary gland volume decreases owing to the regression of the secretory structure. In general, common mammary gland patterns have been shown for both goats and sheep throughout the several lactation stages, although the number of studies is limited. The main objective of this manuscript is to review the colostrogenesis and lactogenesis processes as well as to highlight the mammary gland morphological patterns underlying milk production during the lactation cycle for small ruminants, and to describe potential differences between goats and sheep, hence contributing to a better description of mammary gland development during lactation for these two poorly studied species.