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.     

Environmental Particulate (PM2.5) Augments Stiffness-Induced Alveolar Epithelial Cell Mechanoactivation of Transforming Growth Factor Beta

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor - beta (TGFβ) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.     

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.     

Low temperature delays development of photosystem II activity in winter rye leaves

The ability of leaves to acclimate photosynthetically to low temperature was examined during leaf development in winter rye plants ( Secale cereale L. cv. Puma) grown at 20°C or at 6°C. All leaves grown at 6°C exhibit increased chlorophyll (Chl) levels per leaf area, higher rates of uncoupled, light-saturated photosystem I (PSI) electron transport, and slower increases in photosystem II (PSII) electron transport capacity, when compared with 20°C leaves. The stoiehiometry of PSI and PSII was estimated for each leaf age class by quantifying Chl in elcctrophorctic separations of Chl-protein complexes. The ratio of PSII/PSI electron transport in 20°C leaves is highly correlated with the ratio of core Chl a -proteins associated with PSII (CPa) to those associated with PSI (CP1). In contrast, PSII/PSI electron transport in 6°C leaves is not as well correlated with CPa/CP1 and is related, in part, to the amount and organization of light-harvesting Chl a/b -proteins associated with PSII. CPa/CP1 increases slowly in 6°C leaves, although the ratio of CPa/CP1 in mature 20°C and 6°C leaves is not different. The results suggest that increased PSI activity at low temperature is not related to an increase in the relative proportion of PSI and may reflect, instead, a regulatory change. Photosynthetic acclimation to low environmental temperature involves increased PSI activity in mature leaves shifted to 6°C. In leaves grown entirely at 6°C, however, acclimation includes both increased PSI activity and modifications in the rate of accumlation of PSII and in the organization of LHCII.