Food or edible material: processes – compositions – and products – Processes – Separating a starting material into plural different...
Reexamination Certificate
2000-11-16
2002-05-21
Hendricks, Keith (Department: 1761)
Food or edible material: processes, compositions, and products
Processes
Separating a starting material into plural different...
Reexamination Certificate
active
06391362
ABSTRACT:
FIELD OF THE INVENTION
Milk fat globule membranes are diagnostically, pharmaceutically and nutriceutically useful proteo-lipid compositions, and the present invention provides for their bulk separation from milk.
BACKGROUND OF THE INVENTION
One remarkable feature of milk is the presence of lipid droplets coated with proteo-lipid material, or, as it is usually referred to as the milk fat globule membrane (MFGM). The MFGM is composed of four layers: the thin membrane, possibly derived from intracellular lipovesicles; the protein coat; the lipid bilayer, primarily derived from the apical plasma membrane and possibly secretory vesicle membranes; and the glycocalyx. Milk protein synthesis during lactation is simultaneously accomplished by the intensive synthesis of membrane components needed to replenish their loss by their extrusion from mammary gland secretory cells. In this sense, milk is a unique deposit of biological membranes synthesized inside secretory epithelial cells.
The study of MFGM proteins has received much attention over the years. Many proteins of the MFGM have been well characterized. Some of the genes encoding these proteins have been cloned. Certain proteins of the MFGM are of special interest because of their involvement in important cellular processes. Milk fat globule membrane (MFGM) contains large quantities of the novel proteins and factors that play physiological roles such as inhibiting the growth of cancer cells, increasing the growth and proliferation of normal cells and lowering blood cholesterol. Two specific proteins in the milk fat globule membranes are MDGI/FABP [mammary derived growth inhibitor (MDGI or FABP (fatty acid binding protein)] a 15 kDa protein and BRCA1/BRCA2 (breast ovarian cancer susceptibility protein). BRCA1 is a tumor supressor protein that is also involved in cell signaling, DNA repair and apoptosis.
Several experiments have been conducted to illuminate the physiological mechanism whereby milk fat derived MDGI/FABP inhibits the growth of mammary cells. One of the experiments has proven that MDGI/FABP is in a phosphorylated form when it is present in mammary cells. The MDGI/FABP is phosphorylated on tyrosine. It is believed that the physiological activity of MDGI/FABP is regulated through its phosphorylation by protein kinases. Protein kinase activity exists in the milk fat globule membranes obtained from bovine and human milk.
Other experiments designed to determine the mechanism of MDGI/FABP on mammary cell growth and differentiation have led to the discovery that MDGI/FABP is in close association with the glycoprotein CD36 within the MFGM. CD36 is an abundant protein found in milk fat from cows. It is involved in cellular differentiation, lipid transport and sequestering oxidized fatty acids in milk. The information currently available suggests that the inhibitory action of MDGI/FABP and its synthetic analogs on cell proliferation is manifested through their interaction with the ectodomain of CD36. Moreover, CD36 may be a receptor for MDGI/FABP.
It has been suggested that the variation in fatty acids in adipocytes extracted from premenopausal and post menopausal women can act directly on growth and differentiation of mammary epithelial cells and the variation of CD36 may act either on the transport of fatty acids or on the transduction of the signal responsible for the stimulation of enzymes catalyzing the conversion of fatty acid into different metabolites.
CD 36 expression is elevated in various primary human breast tumors. Diets high in saturated fats may influence the synthesis of CAMs such as CD36, thereby making certain tissues such as the mammary tissue more susceptible to carcinogens.
Unsaturated long chain fatty acids may also regulate levels of CD36. Heart type fatty acid binding protein is increased in mammary tumors, propagated in nude mice, in response to either unsaturated or saturated fatty acids.
It has also been recently demonstrated that two genes, namely, BRCA1 encoding a 220 kDa protein, and BRCA2 encoding an approximate 420 kDa protein, are involved in hereditary breast and ovarian cancers. The BRCA1 protein contains an N-terminus zinc finger domain, i.e. the region which can bind to DNA. The BRCA1 protein may represent one of the transcription factors, playing an important role in the differentiation of mammary gland cells. The C-terminal end of the BRCA1 is essential to normal BRCA1 function in breast epithelial cells, because patients inheriting 1853Stop develop very early onset breast cancer. The development of hereditary breast cancers can be seen as the result of mutations or deletions of the BRCA1 gene leading to the production of altered forms (truncated) of BRCA1 protein which cannot function as suppressors of cell growth (tumor suppressors).
It has been shown that the BRCA1 protein also can be involved in sporadic breast cancers. In this case, the transport of BRCA1 protein into the nucleus of cell is believed to be altered. Therefore, an accumulation of BRCA1 protein occurs in the cytoplasm of mammary gland secretory cells. Although there is some controversy regarding this hypothesis, the result cannot be ignored. It has been reported that the expression of BRCA1 in sporadic cancers is diminished. Quite recently, it was reported that BRCA1 is a secreted protein. Clustered BRCA1 proteins were detected by immunogold electron microscopy in small membrane bound vesicles in the apical cytoplasm of mammary epithelial cells. BRCA1has also been localized in bovine mammary epithelial cells from lactating cows. It is suggested that BRCA1 can manifest its function through the secretion and subsequent binding to the putative receptor of the same cell.
Though there is no consensus on the mechanism of action of BRCA1we have to accept that BRCA1 is an important protein for development and differentiation of mammary gland secretory epithelial cells. Mutations or microdeletions of the BRCA1 gene or altered expression of BRCA1 mRNA and BRCA1 protein can lead to dedifferentiation with possible formation of cancer cells.
Recently, the complete sequence of the BRCA2 gene was reported. This gene encodes the protein of 3418 amino acids i.e. this protein would be about 420 kDas. Biochemical function of BRCA2 is not yet clear though the presence of regulatory signals are indicated. A mutational profile of BRCA2 differs from BRCA1 and is characterized by microdeletions rather than point mutations. The microdeletions in BRCA2 gene would explain the truncated forms of the BRCA2protein. The 15 mutations observed so far by the Myriad group are quite distinct. This situation can complicate the development of the genetic test for the determination of predisposition to breast cancer. BRCA2 as BRCA1 has a sequence (“granin consensus”) which is typical for a number of secretory proteins. The secretion of BRCA2 protein by mammary epithelial cells still has to be determined.
The protein BRCA1 is produced in mammary cells, but isolating BRCA1 from mammary cells is expensive and time consuming, such that it is entirely impractical to isolate commercial quantities of BRCA1. Therefore, it will be extremely important to develop an inexpensive method of isolating BRCA1 if it is to be used as a treatment.
Butyrophilin (Bph) is a 62-67 kDa glycoprotein found in the MFGM. It is the major glycoprotein of the bovine MFGM accounting for over 40 and 50% of the total protein on a weight and molar bases, respectively. Bph is specifically expressed in bovine mammary tissue. Bph is abundant only during lactation in secretory epithelial cells.
Bph has been detected in the apical pole of the secretory epithelial cell during budding of fat droplets. Butyrophilin protein and its transcripts have only been detected in the MFGM and mammary secretory epithelial cell and not in any other tissue or cell studied. It can therefore be considered as an organ specific protein.
The Bph gene is clearly regulated developmentally in the mammary gland with maximal expression during lactation. The amounts of bovine Bph mRNA increase dramatically in the last 6
Hendricks Keith
Webb Ziesenheim & Logsdon Orkin & Hanson, P.C.
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