Lack of functional alpha-lactalbumin prevents involution in Cape fur seals and identifies the protein as an apoptotic milk factor in mammary gland involution

The mammary gland undergoes a sophisticated programme of developmental changes during pregnancy/lactation. However, little is known about processes involving initiation of apoptosis at involution following weaning. We used fur seals as models to study the molecular process of involution as these animals display a unique mammary gland phenotype. Fur seals have long lactation periods whereby mothers cycle between secreting copious quantities of milk for 2 to 3 days suckling pups on land, with trips to sea alone to forage for up to 23 days during which time mammary glands remain active without initiating apoptosis/involution.<br /

TRANSGENIC NONHUMAN MAMMALS PRODUCING FIBRINOGEN IN MILKAND METHODS OF PRODUCING FIBRIN

A transgenic, non-human mammalian animal is capable of expressing a heterologous gene for human or other recombinant physiologically functional fibrinogen holoprotein or individual subunit chain polypeptides thereofora modified or fusion fibrinogen in mammary glands of the animals and secreting the expressed product into a body fluid. Methodol ogy employing such a mammal yields recombinant physiologically functional fibrinogens, subunit chain polypeptides thereof, and modified or fusion fibrinogens

Molecular Biology of an Autocrine Inhibitor of Milk Secretion

Research over many years has shown that the rate of milk secretion is regulated by frequency and completeness of milk removal. The effect of milk removal occurs through local mechanisms within each mammary gland, and recent work indicates that local control is through feedback inhibition by a novel milk protein termed FIL, Feedback Inhibitor of Lactation. Evidence from studies in mammary cell culture suggests that FIL controls the rate of milk secretion, mediating the effect of frequency and completeness of milk removal, by inhibition of constitutive secretion, which involves reversible blockade of mammary membrane trafficking. Due to its effects on membrane trafficking, FIL may also regulate mammary differentiation. This may provide a mechanistic explanation for the developmental changes associated with sustained alterations in milking frequency or efficiency. For example, extended frequent milking elicits a significant increase in secretory cell differentiation as measured by mRNA abundance and activities of key enzymes involved in milk synthesis. However, neither the developmental changes at the level of gene expression or the mechanism underpinning these responses has been characterised in detail. The aim of this project was therefore to investigate whether frequency of milking does indeed control expression of key milk protein genes and to investigate the mechanisms underpinning the putative regulation of gene expression - specifically, to determine if FIL is competent to influence mammary gene expression. In the first phase of the project, manipulation of milking frequency and concomitant changes in the rate of milk secretion were found to be accompanied in the long term, but not in the short term, by changes in milk protein mRNA abundance. Treatments which did not change milk yield did not affect milk protein gene expression, indicating that changes in milk protein gene expression, like changes in milking frequency are dependent on effective manipulation of milk removal. To investigate the molecular mechanisms underpinning the increase in milk protein mRNA abundance, demonstrated in vivo, goat mammary cells m primary culture were treated with milk fractions and FIL to determine if this protein was indeed competent to modulate milk protein gene expression. These studies demonstrated that long term exposure to FIL decreases milk protein mRNA abundance in vitro, lending further credence to the theory that FIL is a regulator of mammary differentiation. Changes in gene expression in response to FIL, demonstrated in vitro, imply that FEL is involved in the developmental response of the gland to frequency of milk removal. Since FIL is itself a mammary gene product, it is also possible that FIL is an autocrine regulator of its own expression. Therefore, the next phase of this project was to clone the gene for FIL, and, if successful investigate the regulation of its gene expression. Several strategies were implemented to clone FII. including screening of goat mammary cDNA libraries with anti-FIL antibody and with synthetic oligonucleotides constructed on the basis of known FIL protein sequence. These strategies were not successful. Whether this was due to library composition, antibody specificity or excessive redundancy in the predicted nucleotide sequence of caprine FIL remains to be determined. In conclusion, the project has shown that the developmental responses to frequency and completeness of milk removal are associated with changes in expression of key milk protein genes, and experiments in cell culture suggest these changes may be elicited by FIL, as a long term consequence of its effects on mammary membrane trafficking

An investigation into the role of autoimmunity in vitiligo

Vitiligo is an acquired depigmenting disorder characterised by the loss of functional melanocytes from the cutaneous epidermis. A role for autoimmunity is supported by the presence of circulating antibodies and T lymphocytes which react against melanocyte antigens in patients with vitiligo. The identification and characterisation of these autoantigens will improve understanding of the immune response in vitiligo, and may allow development of better therapies and diagnostic tools. Candidate immunoregulatory genes may predispose to vitiligo. However, this study failed to find an association between vitiligo and a polymorphism of the cytotoxic T lymphocyte antigen-4 (CTLA-4), although the polymorphism increases the likelihood of autoimmune endocrinopathy patients developing vitiligo. The epitopes on melanocyte-specific antigens tyrosinase and Pmel17 which are recognised by antibodies in vitiligo patient sera were identified by molecular mapping. Multiple regions of tyrosinase and at least two domains on PmeI17 were identified as B cell epitopes. Sequence analysis revealed that the tyrosinase epitopes are likely to be cross-reactive with tyrosinase-related proteins but that the antibody response to Pmel17 is distinct. Antibody reactivity to a melanocyte protein, MelanA, targeted by a cellular immune response in vitiligo and melanoma was investigated by immunoblotting and radioimmunoassay. No MelanA-specific antibodies were isolated suggesting that either it is not a target of the humoral immune response in vitiligo, or that antibody reactivity was not detectable by the methods used. To identify novel vitiligo autoantigens, a melanoma cDNA expression library was constructed in a phage-display cloning system and immunoscreened with vitiligo patient IgG. Several possible autoantigens were enriched by this technique, including proteins previously characterised as autoantigens in other disorders. Additionally, humoral reactivity was identified to a protein with a possible role in pigmentation, the melanin-concentrating hormone receptor 1 (MCHR1). MCHR1-specific antibodies were detected in 16.4% (9/55) of vitiligo patients but not in other diseases or healthy control subjects. The study demonstrates the usefulness of phage-display for further autoantigen identification in vitiligo