Polyunsaturated fatty acids enhance the proliferation of mouse mammary epithelial cells stimulated by epidermal growth factor (EGF) by modulating the post-receptor signaling pathways. The growth stimulatory effect of these fatty acids is completely inhibited by pertussis toxin, whereas the inhibition of EGF and insulin stimulated growth is only partial. The treatment of cell cultures with 12-O-tetradecanoyl-phorbol-13 acetate (TPA) reverses the growth inhibitory effect of pertussis toxin and fully restores the growth as was in the control cultures untreated with the toxin suggesting a role for PKC in this reversal. It appears that the functions of Gi-proteins are required in the mediation of fatty acid effect on growth. The predominant types of Gi alpha in mammary epithelial cells are Gi alpha 1, Gi alpha 2, and Gi alpha 3. Among these, the levels of Gi alpha 1 and 2 appears to be regulated by steroid hormones. Linoleic acid raises the level of GTP-bound Ras in the cells above the levels induced by EGF. Pertussis toxin reduces the level of Ras-GTP and inhibits phosphorylation of MAP kinase by EGF. It has been speculated that Gi-proteins interact with the receptor bound nucleotide exchange factor and the membrane anchored Raf kinase and constitute two sites for pertussis toxin action. The phosphorylation by PKC may uncouple Gi-protein interaction with these effectors and enable the agonist-induced signals to bypass the inhibitory action of PT on growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Expression and purification of functional human alpha-1-Antitrypsin from cultured plant cells.

Human alpha-1-antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon-optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice-derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant-derived AAT for treatments of various AAT-deficient diseases are discussed.

Animals↗

PubMedDate not supplied

Effect of dietary supplementation of fatty acids and vitamins on the breeding performance of the carp Catla catla.

Five isonitrogenous diets (approximately 33% crude protein) were fed to the brood female carp, Catla catla (weighing 3.0 to 5.5 kg), for a period of 93 days in order to observe their breeding performance in earthen ponds. Diet-I (control) contained only basic ingredients like rice bran, ground-nut oil cake, roasted soybean meal, fish meal and mineral mixture; diet-II contained added vitamins; diet-III contained added vitamins and vegetable oil (rich in n-6 polyunsaturated fatty acids, PUFA); diet-IV contained added vitamins and fish oil (rich in n-3 PUFA); and diet-V contained added vitamins and a mixture of vegetable and fish oils. The results showed that nutritional quality of the diet considerably influenced breeding performance in the species. The total number of matured females was the highest in the diet-V group and maturity was advanced by 35 days in this group compared to the control. In diet-III and diet-V groups, all the maturated females bred fully and the relative fecundity was increased significantly in diet III, IV and V. The maximum (73.4%) fertilisation rate was observed in the diet-V group, followed by 61.3%, 56.8%, 49% and 22.7% in diet-I, diet-IV, diet-III and diet-II groups respectively. Most of the eggs in the diet-II treatment group remained immature. The various data thus obtained suggest that dietary supplementation of both n-3 and n-6 PUFA, is essential to improve gonadal maturation, breeding performance and spawn recovery in the Catla female broodstock.

Animal Feed↗

PubMedDate not supplied

Adeno-associated virus vector mediated transduction of primary normal human breast epithelial cells.

Cultured human breast epithelial cells from reduction mammoplasty specimens were transduced using an adeno-associated virus vector encoding the marker gene E. coli -galactosidase. Subconfluent, growing, breast epithelial cells were more easily transduced than confluent, quiescent, cells. Transduction of non-dividing confluent cells could be greatly increased by ultraviolet light-induced DNA damage or by prior exposure to the DNA synthesis inhibitor hydroxyurea. The effects of ultraviolet light and hydroxyurea on transduction were additive when these agents were applied together.

Breast↗

PubMedDate not supplied

Estrogen and progesterone receptors can be maintained in normal human breast epithelial cells in primary culture and after transplantation into nude mice.

The estrogen and progesterone receptor expression was determined in breast epithelial cells from reduction mammoplasty specimens. Only a subset of the luminal epithelial cell population was found to be positive for these receptors. When these cells were subsequently cultured in 3-dimensional collagen gel system (in vitro model) or transplanted into nude mice (in vivo model), the cells retained their ability to express both receptors in these experimental systems. The maintenance of primary normal human breast epithelial cells expressing the estrogen and progesterone receptors in experimental systems has not been reported previously.

Animals↗