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Pharo E.A.,University of Melbourne | Pharo E.A.,Cooperative Research Center for Innovative Dairy Products | Pharo E.A.,Australian Department of Primary Industries and Fisheries
Molecular and Cellular Endocrinology | Year: 2014

Marsupial ELP (early lactation protein) and its eutherian orthologue, CTI (colostrum trypsin inhibitor) are expressed in the mammary gland only for the first 100. days postpartum (Phase 2A) in the tammar wallaby and during the bovine and canine colostrogenesis period 24-36. h postpartum respectively. The factors which regulate temporal ELP and CTI expression are unknown. A tammar mammary gland explant culture model was used to investigate ELP gene regulation during pregnancy and early- and mid-lactation (Phase 1, 2A and 2B respectively). Tammar ELP expression could only be manipulated in explants in vitro if the gene was already expressed in vivo. ELP expression was maximal in Phase 1 explants treated with lactogenic hormones (insulin, hydrocortisone and prolactin), but unlike LGB (β-lactoglobulin), ELP expression was maintained in insulin or insulin and hydrocortisone over a 12-day culture period. In contrast, ELP was down-regulated when cultured without hormones. ELP could not be induced in explants cultured from mid-lactation which suggested that transcriptional repressors may prevent ELP expression during this period. © 2013 Elsevier Ireland Ltd.


Pharo E.A.,University of Melbourne | Pharo E.A.,Cooperative Research Center for Innovative Dairy Products | De Leo A.A.,University of Melbourne | De Leo A.A.,Cooperative Research Center for Innovative Dairy Products | And 9 more authors.
BMC Evolutionary Biology | Year: 2012

Background: The marsupial early lactation protein (ELP) gene is expressed in the mammary gland and the protein is secreted into milk during early lactation (Phase 2A). Mature ELP shares approximately 55.4% similarity with the colostrum-specific bovine colostrum trypsin inhibitor (CTI) protein. Although ELP and CTI both have a single bovine pancreatic trypsin inhibitor (BPTI)-Kunitz domain and are secreted only during the early lactation phases, their evolutionary history is yet to be investigated. Results: Tammar ELP was isolated from a genomic library and the fat-tailed dunnart and Southern koala ELP genes cloned from genomic DNA. The tammar ELP gene was expressed only in the mammary gland during late pregnancy (Phase 1) and early lactation (Phase 2A). The opossum and fat-tailed dunnart ELP and cow CTI transcripts were cloned from RNA isolated from the mammary gland and dog CTI from cells in colostrum. The putative mature ELP and CTI peptides shared 44.6%-62.2% similarity. In silico analyses identified the ELP and CTI genes in the other species examined and provided compelling evidence that they evolved from a common ancestral gene. In addition, whilst the eutherian CTI gene was conserved in the Laurasiatherian orders Carnivora and Cetartiodactyla, it had become a pseudogene in others. These data suggest that bovine CTI may be the ancestral gene of the Artiodactyla-specific, rapidly evolving chromosome 13 pancreatic trypsin inhibitor (PTI), spleen trypsin inhibitor (STI) and the five placenta-specific trophoblast Kunitz domain protein (TKDP1-5) genes. Conclusions: Marsupial ELP and eutherian CTI evolved from an ancestral therian mammal gene before the divergence of marsupials and eutherians between 130 and 160 million years ago. The retention of the ELP gene in marsupials suggests that this early lactation-specific milk protein may have an important role in the immunologically nave young of these species. © 2012 Pharo et al.; licensee BioMed Central Ltd.


Pharo E.A.,University of Melbourne | Pharo E.A.,Cooperative Research Center for Innovative Dairy Products | Cane K.N.,University of Melbourne | Cane K.N.,Cooperative Research Center for Innovative Dairy Products | And 7 more authors.
Gene | Year: 2016

The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48. h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3. months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk. © 2015 Elsevier B.V.


Riley L.G.,University of Sydney | Riley L.G.,Cooperative Research Center for Innovative Dairy Products | Riley L.G.,Genetic Metabolic Disorders Research Unit | Gardiner-Garden M.,Garvan Institute of Medical Research | And 11 more authors.
Animal Genetics | Year: 2010

An in vitro bovine mammosphere model was characterized for use in lactational biology studies using a functional genomics approach. Primary bovine mammary epithelial cells cultured on a basement membrane, Matrigel, formed three-dimensional alveoli-like structures or mammospheres. Gene expression profiling during mammosphere formation by high-density microarray analysis indicated that mammospheres underwent similar molecular and cellular processes to developing alveoli in the mammary gland. Gene expression profiles indicated that genes involved in milk protein and fat biosynthesis were expressed, however, lactose biosynthesis may have been compromised. Investigation of factors influencing mammosphere formation revealed that extracellular matrix (ECM) was responsible for the initiation of this process and that prolactin (Prl) was necessary for high levels of milk protein expression. CSN3 (encoding κ-casein) was the most highly expressed casein gene, followed by CSN1S1 (encoding αS1-casein) and CSN2 (encoding β-casein). Eighteen Prl-responsive genes were identified, including CSN1S1, SOCS2 and CSN2, however, expression of CSN3 was not significantly increased by Prl and CSN1S2 was not expressed at detectable levels in mammospheres. A number of novel Prl responsive genes were identified, including ECM components and genes involved in differentiation and apoptosis. This mammosphere model is a useful model system for functional genomics studies of certain aspects of dairy cattle lactation. © 2009 Stichting International Foundation for Animal Genetics.

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