Glucocorticoid action on the immune system

Glucocorticoids have profound effects on immune function that are mediated, in part, by steroid-induced cell death. Our studies have been aimed at identifying the mechanism of this lymphocytolytic process using the rat thymocyte as a model system. Administration of glucocorticoids in vivo resulted in internucleosomal cleavage of the lymphocyte genome that was detectable within 2 h of treatment and increased with time after hormone administration. Six h after steroid treatment greater than 50% of the genome was degraded, yet cell viability remained greater than 90% indicating that this event preceded cell death. Furthermore, this process appeared to be mediated by the glucocorticoid receptor since the antagonist RU 486 blocked glucocorticoid-mediated DNA degradation. To further characterize this lymphocytolysis we have analyzed glucocorticoid-treated thymocytes for nucleases. Two families of nuclear proteins have been identified, a 30-32 kDa doublet and a series of 3-4 proteins that are 12-19 kDa, both of which are induced by glucocorticoid treatment (137 +/- 6% and 342 +/- 24%, respectively) and have prominent nuclease activity. These nucleases can also be induced in vitro indicating that glucocorticoids act directly on thymocytes to mediate this response. Moreover, this nuclease induction, like glucocorticoid-mediated DNA degradation, could be blocked by RU 486. Based on these findings we propose a working model of glucocorticoid-mediated lymphocytolysis in which these steroids, acting via a receptor mediated process, induce the expression of a lysis gene product (nuclease) which degrades the genome and results in cell death.     

Changes in cellular gene expression in rat thyroid epithelial cells transformed by the Kirsten murine sarcoma virus

We have exploited a recently characterized system of rat thyroid epithelial cells transformed by the wild-type (wt) and a temperature-sensitive (ts) mutant strain of the Kirsten murine sarcoma virus (Ki-MSV) in order to study the effects of the K-ras oncogene on the gene expression of differentiated thyroid epithelial cells. By using cDNAs isolated from normal thyroid glands as probes, we were able to identify three sets of cellular sequences whose expression is influenced by the v-K-ras oncogene. The first set of genes is irreversibly repressed by transformation with both the wt and the ts viruses. The second set of genes is repressed in the ts-Ki-MSV-transformed cells but not in the same cells grown at the nonpermissive temperature. A third set of genes is present at higher levels at the nonpermissive temperature than at the permissive temperature. This system has allowed us to isolate and characterize a number of cDNA clones belonging to each of these three sets of genes. These specific cDNAs are suitable probes to study phenotypical changes during transformation of epithelial cells.     

Purification and characterization of the beta-adrenergic receptor kinase

The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues. The kinase can be purified from bovine cerebral cortex to greater than 90% homogeneity by sequential chromatography on Ultrogel AcA34, DEAE-Sephacel, CM-Fractogel, and hydroxylapatite. This results in an approximately 20,000-fold purification with an overall recovery of 12%. The purified kinase has an Mr approximately 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several findings indicate that this peptide contains the beta-ARK activity. First, on hydroxylapatite chromatography the enzyme activity coelutes with the Mr approximately 80,000 protein as revealed by Coomassie-Blue staining. Second, under phosphorylating conditions the Mr approximately 80,000 protein is phosphorylated. Finally, the Mr approximately 80,000 protein specifically interacts with reconstituted agonist-occupied beta-AR. Kinetic parameters of the enzyme for beta-AR are Km = 0.25 microM and Vmax = 78 nmol/min/mg whereas for rhodopsin the values are Km = 6 microM and Vmax = 72 nmol/min/mg. The Km value of the enzyme for ATP is approximately 35 microM using either beta-AR or rhodopsin as substrate. Receptor phosphorylation by beta-ARK is effectively inhibited by Zn2+, digitonin and a variety of salts. The availability of purified beta-ARK should greatly facilitate studies of its role in receptor desensitization.     

Factors responsible for the differences in cultural estimates and direct microscopical counts of populations of bacterivorous nanoflagellates

Bacterivorous nanoflagellates (microflagellates) have been routinely enumerated in marine and freshwater samples using either a Most Probable Number (MPN) culture method or by a direct microscopical counting method (DC). These two techniques typically yield highly disparate estimates of the density of nanoflagellates in natural samples. We compared these methods with seawater and marine snow (macroscopic detrital aggregate) samples collected from surface waters throughout the North Atlantic and in freshwater samples collected at three stations in Lake Ontario. Densities of nanoflagellates determined by the two methods differed by as much as four orders of magnitude; the MPN estimate rarely exceeded 10% of the microscopical count, and averaged 1% of this count. The MPN estimate constituted a higher percentage of the DC value in environments with high concentrations of nanoflagellates relative to environments with low concentrations of nanoflagellates. The ratio of the culture count to the microscopical count (MPNDC) increased along an environmental gradient from oligotrophy to eutrophy, and was positively correlated with the density of bacteria in the samples. In laboratory experiments with two species of bacterivorous nanoflagellates, the MPN count constituted a much greater percentage of the DC count during the exponential growth phase of the nanoflagellate than during the stationary growth phase. Differences in the estimates of nanoflagellate density obtained with these two techniques probably can be explained by the trophic mode of these protozoa, their growth stage, and the amenability of these species to laboratory culture.     

Genetic convergence during serial in vitro passage of a polyclonal squamous cell carcinoma

A cell line was established from an in situ squamous cell carcinoma of the skin (Bowen's disease), and its in vitro karyotypic evolution was cytogenetically analyzed. Initially, considerable genetic heterogeneity was evident. Nine cytogenetically abnormal clones, eight of which were apparently unrelated, were found among the 83 metaphases analyzed from the primary culture and the first passage. With increasing time in culture this complexity was reduced, so that a single clone dominated passages 7-11. The clone that emerged from this genetic convergence had a t(12;17)(p13;q21) as the sole abnormality. Our findings indicate that the cytogenetic multiclonality that has been repeatedly detected in short-term cultures of squamous cell carcinomas is not caused by the in vitro conditions. Instead, the principles of Darwinian selection apply: the altered, but stable, selection pressure facing a newly established and initially multiclonal cell line will lead to a reduction of genetic heterogeneity until the one clone that now has the proliferative advantage outgrows the other subpopulations.     

Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization

Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.     

The rise in testicular androgens during the first days of treatment with an LHRH agonist in the dog can be blocked by aminoglutethimide or ketoconazole

Up to day 6 of treatment of adult dogs, daily subcutaneous administration of 50 micrograms of the LHRH agonist [D-Trp6, des-Gly-NH2-10]LHRH ethylamide causes up to a 3-fold increase in serum testosterone (T) concentration which is followed by a progressive decrease to castration levels (less than or equal to 0.2 ng/ml) at later time intervals (up to 21 days, the last time interval studied). Both aminoglutethimide and ketoconazole, two inhibitors of steroid biosynthesis, cause a 30-40% rise in serum T when administered alone. However, either drug administered in combination with the LHRH agonist completely blocks the transient rise in serum T observed when the LHRH agonist is administered alone. On the other hand, the LHRH agonist prevents the secondary rise in steroid secretion observed when either of the two inhibitors of steroid secretion is used alone. Administration of the pure antiandrogen Flutamide alone or in combination with LHRH-A and an inhibitor of steroid biosynthesis does not influence serum T levels. When the serum levels of pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone, dehydroepiandrosterone (DHEA), androstenedione (delta 4-dione), androst-5-ene-3 beta, 17 beta-diol (delta 5-diol), T, dihydrotestosterone (DHT), androstane-3 alpha, 17 beta-diol, androstane-3 beta. 17 beta-diol and 17 beta-estradiol (E2) are analyzed in detail, it can be seen that both aminoglutethimide and ketoconazole not only prevent the rise in serum steroids observed during the first 8 days of treatment with the LHRH agonist but that both compounds enhance the inhibitory effect of the LHRH agonist at later time intervals. A predominant inhibitory effect of ketoconazole is exerted on 17,20-desmolase activity. Aminoglutethimide has little influence on the loss of serum LH bioactivity induced by the LHRH agonist while ketoconazole stimulates the concentration of serum bioactive LH in the absence or presence of simultaneous treatment with the LHRH agonist. The present data clearly demonstrate that aminoglutethimide or ketoconazole can prevent the rise in serum androgens accompanying the first days of treatment with an LHRH agonist in the dog. Moreover, after 3 weeks of treatment, the inhibitory effect of the LHRH agonist on serum androgen levels is enhanced by addition of aminoglutethimide or ketoconazole. Moreover, Flutamide does not interfere with the inhibitory action of the LHRH agonist, aminoglutethimide or ketoconazole, thus suggesting that maximal inhibition of androgen action is likely to be achieved by a combination of these drugs.     

Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.     

Rainbow trout p53: cDNA cloning and biochemical characterization.

We have cloned and sequenced the p53-encoding cDNA of rainbow trout (Salmo gairdneri). The encoded product contains the characteristics found in all p53 proteins: (i) the five highly conserved domains, (ii) an acidic N terminus, (iii) a hydrophilic C terminus, and (iv) a penultimate serine residue. Furthermore, we demonstrate that the rainbow trout p53 is able to specifically interact with the SV40 large T antigen.     

Physiological Responses of Phytoflagellates to Dissolved Organic Substrate Additions. 2. Dominant Role of Autotrophic Nutrition in Pyrenomonas salina (Cryptophyceae)

The enhancement of algal growth by organic substrate assimilationis a common laboratory observation, yet few studies have addressedthe interaction of dissolved organic compounds and environmentalfactors for controlling the relative contribution of heterotrophyand autotrophy to the nutrition of these algae. The effectsof light intensity and glycerol addition on the growth, cellvolume, pigmentation, and carbon uptake of the facultative heterotroph,Pyrenomonas salina Santore, were examined. Glycerol additionto cultures growing at a limiting light intensity increasedthe growth rate, increased the average cell volume and cellularstarch content, decreased the cellular phycoerythrin to chlorophyll ratio, and had no effect on the CO₂ fixation rate cell^–1.Glycerol addition to cultures growing at a moderate light intensitythat was saturating for photo-autotrophic growth increased theaverage cell volume and cellular starch content but had no effecton the CO₂ fixation rate cell^–1. The results indicatethat autotrophy was the major process for carbon acquisitionduring the growth of P. salina, but that carbon acquisitionfrom glycerol catabolism also was used to partially supportgrowth of the alga at the limiting light intensity. In addition,glycerol presumably was used to fulfill the energy and/or reductantrequirements of the alga, and to increase the reserve carbohydrate(starch). ¹ Current address and address for correspondences: Horn PointEnvironmental Laboratories, University of Maryland, PO Box 775,Cambridge, Maryland 21613, U.S.A. (Received October 29, 1990; Accepted May 31, 1991)