Mechanisms of glucocorticoid function in human leukemic cells: analysis of receptor gene mutants of the activation-labile type using the covalent affinity ligand dexamethasone mesylate

In the cultured acute lymphoblastic leukemic (ALL) cell line, clones of sensitive cells are killed by receptor-occupying concentrations of glucocorticoids. In addition, several types of resistance have been identified. The types of resistance are r- (glucocorticoid binding site loss), ract/l (activation labile receptors) and r+ly- (defective lysis mechanism). The two types of receptor mutants have been examined for the presence and expression of the glucocorticoid receptor (GR) gene. Southern blot analysis, using a full-length cDNA probe for human GR, shows that the gene in both is grossly intact. Examination of the expression of the gene by Northern blots reveals the presence of normal, 7-kb message in both types of receptor mutants, though in amounts somewhat reduced from wild-type. This report focuses on the activation labile mutants. Since characterization of these mutants suggests that they can bind ligand but not retain it during activation, we hypothesized that they would respond normally to a ligand that could not be lost during activation. This seems to be the case. When the covalent affinity ligand dexamethasone mesylate, itself a partial glucocorticoid agonist/antagonist, is used, the ract/l cells are killed to an extent corresponding to that evoked by a sub-optimal concentration of the full agonist dexamethasone. We conclude: (1) that the ract/l receptors can function to kill cells if provided a ligand that they do not lose during activation; (2) that the partial agonist activity of dexamethasone mesylate for cell killing is not due to release of a small amount of free dexamethasone; (3) that the poor agonist activity of dexamethasone mesylate receptor complexes suggests that the role of steroid is strictly to participate in conversion of the receptor to its DNA binding form, after which presence of the steroid actually interferes with proper receptor action.     

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.     

Contrasting effects of oestradiol-17 beta and human chorionic gonadotrophin on steroidogenesis in the rabbit corpus luteum

On Day 10 of pseudopregnancy, rabbits were given an i.v. injection of hCG (10-20 i.u.) that was sufficient to cause new ovulations and the loss of follicular oestradiol secretion. There was an immediate 3-4-fold rise in serum progesterone which returned to near prestimulation values (approximately 27 ng/ml) within 12 h in the presence of an implant containing oestradiol-17 beta. In the absence of oestradiol, serum progesterone continued to decline to reach low values (approximately 4 ng/ml) within 24 h and the original corpora lutea subsequently regressed. The administration of oestradiol 24 h after injection of hCG, when progesterone secretion was low, arrested any further decline in progesterone and then restored serum progesterone to normal values. This steroidogenic effect of oestradiol in vivo was a function of enhanced luteal steroidogenesis; corpora lutea removed and incubated for 12 h produced progesterone at high, linear rates, whereas the corpora lutea from animals that did not receive oestradiol produced low or insignificant quantities of progesterone in vitro. We conclude that hCG at these doses is compatible with continued responsiveness of the corpora lutea to oestrogen and that hCG produces its luteolytic effect primarily by ovulating follicles, thus stopping the secretion of the luteotrophic hormone, oestradiol.     

Human erythrocyte proteins associated with adenosine 3',5'-cyclic monophosphate action.

Two adenosine 3',5'-cyclic monophosphate (cyclic-AMP)-binding protein factors (molecular weight 230,000) have been partially purified from human erythrocytes. One of these proteins seems to be different from the cyclic-AMP-binding component of the cyclic-AMP-dependent protein kinases. These protein factors are also capable of binding adenosine. We present data also on two forms of cyclic-AMP-dependent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) partially purified from the cytosol of normal human erythrocytes. Kinase I has been classified as type I enzyme on the basis of its activation when preincubated with protamine, histone or NaCl. The substrate specificities of the two kinases and many of their kinetic parameters are rather similar. Their subunit structure is reminiscent of that of kinases obtained from other sources. The catalytic subunit of both enzymes reversibly cross-react with the regulatory subunit of kinase I from the rabbit red blood cell.     

Molecular Targets for the Testing of COVID‐19

The pandemic outbreaks of coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), spread all over the world in a short period of time. Efficient identification of the infection by SARS‐CoV‐2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with the infectious disease. In Taiwan, a COVID‐19 Open Science Platform adheres to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid‐February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID‐19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS‐CoV‐2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID‐19 virus.     

Identification and Characterization of Pothos latent virus Causing Necrotic Ringspots and Line Patterns on Lisianthus (Eustoma grandiflorum) in Taiwan

Lisianthus (Eustoma grandiflorum) grown in screenhouses in Taiwan showed ringspots and concentric line patterns on leaves. A virus having isometric particles approximately 30–32 nm in diameter was isolated from affected lisianthus. Combined results of biological, cytological, serological, molecular and phylogenetic analyses show that the virus can be identified as Pothos latent virus (PoLV), genus Aureusvirus, family Tombusviridae. Inoculating the virus on non‐infected lisianthus plants reproduced the symptoms previously observed in the field. So, this is the first report of PoLV causing disease in lisianthus and the first report of the virus in Taiwan.     

Correct Expression of spec2a in the sea urchin embryo requires both Otx and other cis-regulatory elements

Strongylocentrotus purpuratus Otx (SpOtx) is required simultaneously in sea urchin development for the activation of endo16 in the vegetal plate and for the activation of spec2a in the aboral ectoderm. Because Otx binding sites alone do not appear to be responsible for the spatially restricted expression of spec2a, additional DNA elements were sought. We show here that consensus Otx binding sites fused to basal promoters are sufficient to activate CAT reporter gene expression in all cell types, although expression in endomesoderm progenitors is enhanced. On the other hand, three non-Otx elements derived from the spec2a enhancer are needed together with Otx sites for specifically aboral ectoderm expression. A DNA element termed Y/CBF, lying just downstream from an Otx site within the spec2a enhancer, mediates general activation in the ectoderm. A second element lying between the Otx and Y/CBF sites, called OER, functions to prevent expression in the oral ectoderm. A third site, called ENR, overlapping another Otx site, is required to repress endoderm expression. Three distinct DNA binding proteins interact sequence specifically at the Y/CBF, OER, and ENR elements. The spec2a enhancer thus consists of closely linked activator and repressor elements that function collectively to cause expression of the spec2a gene in the aboral ectoderm.     

Dendritic development: lessons from Drosophila and related branches

Dendrites show remarkable diversity in morphology and function, but the mechanisms that produce the characteristic forms is poorly understood. Insect systems offer a unique opportunity to manipulate and study identified neurons in otherwise undisturbed environments. Recent studies in Drosophila show that dendritic targeting, branching patterns, territories, and metamorphic remodeling are controlled in specific ways, by intrinsic genetic programs and extrinsic cues, with important implications for function. Here, we review some recent advances in our understanding of dendritic development in insects, focusing primarily on insights that have been gained from studies of Drosophila.     

Nanos and Pumilio are essential for dendrite morphogenesis in Drosophila peripheral neurons

Much attention has focused on dendritic translational regulation of neuronal signaling and plasticity. For example, long-term memory in adult Drosophila requires Pumilio (Pum), an RNA binding protein that interacts with the RNA binding protein Nanos (Nos) to form a localized translation repression complex essential for anterior-posterior body patterning in early embryogenesis. Whether dendrite morphogenesis requires similar translational regulation is unknown. Here we report that nos and pum control the elaboration of high-order dendritic branches of class III and IV, but not class I and II, dendritic arborization (da) neurons. Analogous to their function in body patterning, nos and pum require each other to control dendrite morphogenesis, a process likely to involve translational regulation of nos itself. The control of dendrite morphogenesis by Nos/Pum, however, does not require hunchback, which is essential for body patterning. Interestingly, Nos protein is localized to RNA granules in the dendrites of da neurons, raising the possibility that the Nos/Pum translation repression complex operates in dendrites. This work serves as an entry point for future studies of dendritic translational control of dendrite morphogenesis.