A simple and fast triplex-PCR for the identification of milk's animal origin in Halloumi cheese and yoghurt.

In this research communication we describe a straightforward triplex-PCR protocol able to differentiate the origin of milk from three closely related species (goat, sheep and cow) in Halloumi, a cheese with Protected Designation of Origin (PDO), and yogurts. Halloumi must contain at least 51% sheep or goat milk, therefore, the fraudulent adulteration of this cheese with excess of cow milk must be routinely tested. The assay employs one universal forward primer and three species-specific reverse primers giving rise to 287 bp (cow), 313 bp (goat), and 336 bp (sheep) amplicons, under the same amplification conditions. This protocol, when used to test a small number of Cyprus commercial products, correctly detected mislabeling in Halloumi (2 out of 6 samples were adulterated) and yogurt brands (1 out of 4 was adulterated). The suggested protocol is a reliable tool for identifying the origin of milk in Halloumi cheeses and yogurts and can be used in any laboratory equipped with a thermocycler and an agarose gel electrophoresis apparatus.

Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2.

BACKGROUND: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2. METHODS: Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation. RESULTS: Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels. CONCLUSION: Our results indicate the probable involvement of p57KIP2 on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.

Restoration of protein synthesis in pancreatic cancer cells by trans-splicing ribozymes.

This report describes the use of trans-splicing ribozymes to restore p16 protein synthesis in pancreatic cancer cells. A group I intron ribozyme was designed to trans-splice the 2 base-deleted p16 transcripts with the wild-type sequence in a pancreatic cancer cell line, which originally produced no p16. Following transfection of the ribozyme construct in AsPC-1 cells, mutant p16 mRNA molecules were repaired and p16 protein synthesis restored. Moreover, these cells exhibited a reduced ability to grow, compared to the untransfected cells. The technology of ribozymes offers an advantage over gene replacement therapy because it maintains the cellular regulation of gene expression. These results indicate that group I intron ribozymes might prove useful towards the therapy of pancreatic cancer and in conjunction with the advancement of powerful delivery systems this technology will play a major role in the therapy of many diseases.