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.     

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.     

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)     

Fluorescence studies on aged and young erythrocyte populations

Structural changes in red blood cell (RBC) membrane are investigated by fluorescence techniques. Results obtained with three probes (DPH, 3-PM and fluorescamine) indicate a significant increase in membrane rigidity associated with aging of RBCs. Discrepancies between our observations and published data could arise from utilization of experimental conditions closer to physiological conditions in our study. Use of intact RBCs continuously manipulated in a 37 degrees C environment could represent experimental conditions favourable to the identification of rheologic membrane changes in senescent RBCs.     

Agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland

The mechanism of agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe (IL) of the rat pituitary gland was investigated. Exposure of neurointermediate lobe to 60 microM (-)apomorphine (APO) for 60 min altered the binding of [125I]-N-(p-aminophenethyl)spiperone (NAPS), a D-2 receptor-specific ligand. The capacity of the tissue to bind the ligand (Bmax) was not significantly altered by the exposure to (-)APO but the affinity for [125I]NAPS was decreased 3.6-fold in (-)APO-exposed tissue. The molar potency of YM-09151-2, a D-2 receptor-specific antagonist, showed a minimal difference between in control and (-)-APO-exposed tissue. However, the molar potency of (-)APO towards the D-2 receptor was diminished. The loss of [125I]NAPS binding in (-)APO-exposed tissue was reversed by the addition of guanyl nucleotide. These data suggest that exposure to agonist causes a persistent occupancy of the high affinity state of the receptor. Exposure to (-)APO had no effect on either basal or forskolin-activated adenylate cyclase activity of the intermediate lobe. However, the inhibitory effect of (-)APO upon adenylate cyclase activity of IL homogenates was diminished when the tissue was exposed to (-)APO before homogenization. Furthermore, the ability of GTP but not 5'-guanylyl imidodiphosphate [Gpp(NH)p] to inhibit enzyme activity diminished in the (-)APO-exposed tissue. These data suggest that an agonist-induced desensitization of D-2 receptor in rat IL is thought to occur by uncoupling the receptor from the inhibitory guanyl nucleotide binding protein (Gi) or potentiating the hydrolysis of GTP by Gi.