Progesterone metabolism in T47Dco human breast cancer cells--II. Intracellular metabolic path of progesterone and synthetic progestins

We show here that progesterone added to the medium of proliferating T47Dco human breast cancer cells is metabolized with a half life of 2-4h. The final metabolic product, 5 alpha-pregnan-3 beta,6 alpha-diol-20-one, (P-metabolite) is released into the medium. This structure suggested that the intracellular metabolism of progesterone involves the enzymes 5 alpha-reductase, 3 beta-hydroxysteroid dehydrogenase, and 6 alpha-hydroxylase. To investigate this pathway, the cells were incubated with a variety of potential substrates. In addition to progesterone, only precursors with the 5 alpha-configuration served as substrates for the enzymes leading to P-metabolite formation. Some precursors with a 5 beta-configuration were also metabolized by T47Dco cells. This metabolism reflected activity by either 3 beta-hydroxysteroid dehydrogenase and/or 6 alpha-hydroxylase but, in contrast to progesterone metabolism, the rates were different and the products were often mixtures. In T47Dco and MCF-7 human breast tumor cells, the reduction at C-3 followed by 6 alpha-hydroxylation, appear to be the major, and possibly only, route of progesterone metabolism. In contrast, preliminary data suggest that in normal human breast epithelial cells, this is not an exclusive route. Androgens are partially subject to the same metabolic enzymes, but synthetic progestins are not metabolized by T47Dco during an 18 h incubation.     

Hymenolepis diminuta: Worm loss and worm weight loss in long-term infections of the rat

Infections of one and two Hymenolepis diminuta established in newly weaned rats continued to grow for the duration of the experiment (238 days), whereas infections of 5 worms per rat became asymptotic around Day 55 postinfection and remained at or below this level thereafter as shown by biomass and mean weight per worm measurements. Infections of 50 worms established in newly weaned rats became asymptotic around Day 28 postinfection and thereafter worms were lost from the rats. Initially the biomass fell with the loss of worms, but by Day 56 a new lower biomass persisted for the remainder of the infection period. This level was maintained, despite diminishing numbers of worms, due to the growth of surviving individuals to a weight exceeding the original weight at maturity by a factor of more than 2. Experiments using rats that were mature at the time of infection demonstrated that the same response occurred, but approximately 3 weeks earlier.     

Immunochemical detection of unique proteolytic fragments of the chick 1,25-dihydroxyvitamin D3 receptor. Distinct 20-kDa DNA-binding and 45-kDa hormone-binding species

We have characterized proteolytic fragments of the chick intestinal 1,25-dihyroxyvitamin D3 (1,25-(OH)2D3) receptor, produced through either exogenous or endogenous protease action, utilizing a variety of physical and functional assays coupled to immunoblot detection methodology. Treatment of intestinal cytosol with increasing concentrations of trypsin resulted in a progressive diminishment of the 60-kDa receptor concomitant with the appearance of a 20-kDa fragment reactive by Western blot analysis to an anti-1,25-(OH)2D3 receptor monoclonal antibody. Cleveland analysis supported the receptor-origin of this 20-kDa fragment: a common immunoreactive species of 12 kDa could be generated by Staphylococcus aureus V8 protease treatment of the intact 60-kDa receptor as well as the 20-kDa proteolytic product. The 20-kDa fragment did not bind hormone but was capable of interacting with DNA-cellulose in a fashion identical to that of the 60-kDa receptor and, therefore, may contain the functional DNA-binding domain of the chick 1,25-(OH)2D3 receptor. Thus, this fragment likely represents the complement of a larger hormone-bound fragment that we have previously described (Allegretto, E. A., and Pike, J.W. (1985) J. Biol. Chem. 260, 10139-10145). In contrast to the exogenous effect of trypsin, incubation of cytosol resulted in the time-dependent formation of an endogenous protease-derived fragment of 45 kDa. Cleveland analysis was consistent with the 60-kDa receptor derivation of the 45-kDa fragment. This species retained the hormone-binding site and the antibody determinant but was devoid of DNA-binding activity. Moreover, it generated neither the trypsin-dependent 20-kDa fragment nor the V8 protease-dependent 12-kDa species and, therefore, was derived from the opposite end of the receptor molecule. These data have facilitated the construction of a schematic model of the chick receptor in which the immunoreactive epitope is located between the functional domains for hormone binding and DNA binding.     

Characterization of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in human neutrophils

Phosphodiesteric cleavage of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) is required for transmembrane signaling by chemoattractants in human polymorphonuclear leukocytes (PMN). Considering the importance of PtdIns-4,5-P2 as a reservoir for second messenger substances, we have characterized the enzyme system that synthesizes this phospholipid in human PMN, consisting of kinases for phosphatidylinositol (PtdIns) and phosphatidylinositol-4-phosphate (PtdIns-4-P). The preferred phosphate donor for both enzymes was ATP as compared with GTP. The respective Km for ATP for PtdIns kinase and PtdIns-P kinase were 0.049 +/- 0.013 and 0.062 +/- 0.005 mM and for GTP were 0.242 +/- 0.016 and 0.186 +/- 0.037 mM. PtdIns stimulated the activity of PtdIns kinase to a greater extent than PtdIns-4-P kinase. PtdIns-4-P inhibited the activity of detergent-solubilized PtdIns kinase and stimulated particulate PtdIns-4-P kinase, whereas both enzymes exhibited substrate inhibition to PtdIns-4,5-P2. Mg2+ was the preferred cation for both enzymes, but the apparent Km values (4.1 +/- 0.9 mM for PtdIns kinase and 1.0 +/- 0.7 mM for PtdIns-4-P kinase) were significantly different (p less than 0.005). Mn2+ partially substituted for Mg2+, and both enzymes were inhibited by Ca2+. The polyamine spermine stimulated PtdIns-4-P kinase activity to a greater extent and at lower concentrations than PtdIns kinase. PtdIns kinase was easily solubilized in both Triton X-100 and Nonidet P-40, whereas PtdIns-4-P kinase remained in a detergent-nonextractable membrane fraction. These findings demonstrate that the enzyme system in human PMN that forms PtdIns-4,5-P2 is composed of two distinct enzymes with similar characteristics.     

Interferon-beta 2/interleukin 6 is a co-stimulant for human T lymphocytes

IFN-beta 2/IL-6 promotes the proliferation of human peripheral blood T cells in the presence of either PHA or Con A. This effect is observed with highly purified T cells and is masked by the addition of as few as 2% monocytes, suggesting that accessory cells are not required and that IFN-beta 2/IL-6 acts directly on the T cells. Both T4-positive and T8-positive cells respond to IFN-beta 2/IL-6 plus PHA. Studies of the time requirements of IFN-beta 2/IL-6 and of PHA in the response of T cells show that optimal co-stimulation occurs when both IFN-beta 2/IL-6 and PHA are added together at the outset of culture, suggesting that IFN-beta 2/IL-6 acts predominantly on resting T cells. Unlike T cell proliferation induced by IL-2, T cell proliferation induced by IFN-beta 2/IL-6 is not blocked by antibodies to the IL-2 R, suggesting that IFN-beta 2/IL-6 does not act by stimulating IL-2 production. Thus, in addition to its previously reported properties, IFN-beta 2/IL-6 stimulates T cells in the presence of mitogen, and may prove to be of considerable importance in the physiologic activation of T cells.     

New host and locality records for two protozoan (Myxosporea, Coccidia) parasites of marine fishes

This is the first report of the myxosporean Ortholinea orientalis from Atlantic herring Clupea harrengus. It infects the kidney tubules and previously was known from Pacific herring Clupea pallasii and navaga Eleginus navaga in the White Sea and North Pacific Ocean. This is also the first report of the coccidian Eimeria raibauti from Norway pout Trisopterus esmarkii. It infects the epithelium of the pyloric ceca and previously was known only from poorcod Trisopterus minutus in the Mediterranean Sea. The new records are both from the northern North Sea.     

Pathfinding by zebrafish motoneurons in the absence of normal pioneer axons.

Individually identified primary motoneurons of the zebrafish embryo pioneer cell-specific peripheral motor nerves. Later, the growth cones of secondary motoneurons extend along pathways pioneered by primary motor axons. To learn whether primary motor axons are required for pathway navigation by secondary motoneurons, we ablated primary motoneurons and examined subsequent pathfinding by the growth cones of secondary motoneurons. We found that ablation of the primary motoneuron that pioneers the ventral nerve delayed ventral nerve formation, but a normal-appearing nerve eventually formed. Therefore, the secondary motoneurons that extend axons in the ventral nerve were able to pioneer that pathway in the absence of the pathway-specific primary motoneuron. In contrast, in the absence of the primary motoneuron that normally pioneers the dorsal nerve, secondary motoneurons did not pioneer a nerve in the normal location, instead they formed dorsal nerves in an atypical position. This difference in the ability of these two groups of motoneurons to pioneer their normal pathways suggests that the guidance rules followed by their growth cones may be very different. Furthermore, the observation that the atypical dorsal nerves formed in a consistent incorrect location suggests that the growth cones of the secondary motoneurons that extend dorsally make hierarchical pathway choices.     

Tumor promoter enhances mitogenesis by PDGF with little effect on PDGF binding

Preincubation of Swiss 3T3 cells with the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) at 37 degrees C is observed to cause only a small (approximately 10%) decrease in maximal binding of 125I-platelet-derived growth factor (125I-PDGF), and does not affect the affinity of 125I-PDGF binding to these cells. Under the same conditions, the affinity of the epidermal growth factor receptor is greatly reduced, possibly resulting from phosphorylation by protein kinase C. TPA is also shown to have no effect on the kinetics of internalization or degradation of bound 125I-PDGF. Although TPA has little or no effect on these properties of the PDGF receptor, it was found to act in a synergistic fashion with low, but not high, concentrations of PDGF to increase DNA synthesis by 3T3 cells. Since TPA has previously been shown to activate protein kinase C, these findings suggest that protein kinase C does not regulate the ligand-binding properties of the PDGF receptor, and that the observed synergism between TPA and PDGF in stimulating mitogenesis reflects effects of TPA on other processes in the mitogenic pathway.