On the mechanism of action of dexamethasone in a rat mast cell line (RBL-2H3 cells). Evidence for altered coupling of receptors and G-proteins

Prolonged exposure of rat basophilic leukemia (RBL-2H3) cells, a cultured analog of rat mast cells, to 0.1 microM dexamethasone resulted in global suppression of various stimulatory events in response to Ag and a global enhancement of the same stimulatory events to the adenosine analog, N-(ethylcarboxamide)adenosine (NECA). We had previously shown that Ag and NECA both activate phospholipase C but by different mechanisms; cells that had been treated with cholera or pertussis toxin, for example, responded to Ag but not to NECA with the release of inositol phosphates, increase in levels of cytosolic Ca2+, and secretion. Because the toxins still inhibited the responses to NECA in dexamethasone-treated cells, the effects of dexamethasone may have been exerted at the level of receptor/G-protein coupling rather than at the level of effector systems. Additional evidence for this was the following: 1) NECA-induced hydrolysis of the inositol phospholipids was still enhanced after permeabilizing (with streptolysin O or Staphylococcus alpha-toxin) and washing the cells; 2) the response to the G-protein stimulant, guanosine 5'-(3-O-thio)triphosphate was also enhanced in permeabilized, dexamethasone-treated cells and 3) binding and kinetic studies suggested that the enhanced responsiveness to NECA was attributable in part to an increase in receptor number. The suppressive action of dexamethasone on Ag-induced hydrolysis of inositol phospholipids, however, was readily lost by permeabilizing RBL-2H3 cells. The results indicate, therefore, that treatment with dexamethasone leads to changes in receptor-coupling mechanisms that are either resistant to (i.e., NECA-mediated responses) or reversed by (i.e., Ag-mediated responses) cell permeabilization.     

Effect of cooling rate,cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min^-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with –30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at – 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and – 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.     

Electron Paramagnetic Resonance and Spin Trapping Study of Radicals Formed During Reaction of Aromatic Amines with isoAmyl Nitrite Under Aprotic Conditions

Diazotization of primary aromatic amines with isoamyl nitrite in benzene at room temperature was studied employing EPR and spin trapping techniques. Nitrosodurene (ND). 2-methyl-2-nitrosopropane (MNP). and 5,5-dimethyl-pyrroline N-oxide (DMPO) were used as spin trapping agents. Aryl radicals were detected employing ND and MNP. Using DMPO as a spin trap most of the amines produced EPR spectra ascribed to adducts with aniline-type radicals (N-centred radicals). The assignments were verified using ¹⁵JN-labeled anilines. Similar spectra of DMPO adducts were recorded from amines treated with benzoyl peroxide or benzophenone plus UV. Possible mechanisms of formation of these adducts (radical trapping versus nucleophilic addition to DMPO followed by oxidation) during treatment of the amines with isoamyl nitrite are discussed.     

Immunological Characterization and Transmembrane Topology of 5-Hydroxytryptamine3 Receptors by Functional Epitope Tagging

Abstract: 5-Hydroxytryptamine₃ (5-HT₃) receptors are the only known monoamine receptors mediating fast excitatory responses in mammalian neurons. Their primary structure as well as their electrophysiological and pharmacological properties show a phylogenetic relation to nicotinic acetylcholine, GABA_A, and glycine receptors. As a prototypical member of this gene superfamily, we investigated the membrane topology of functional homomeric 5-HT₃ receptors by using epitope tagging of the channel subunits expressed in heterologous systems. Visualization of 5-HT₃ receptors in transfected COS-7 cells, either in western blot (molecular mass 61.2 ± 0.8 kDa) or in situ, was performed with previously characterized antibodies recognizing artificial epitopes as well as with anti-fusion protein antibodies directed against a wild-type receptor intracellular domain. The extracellular location of the distal C-terminal tagged domain demonstrates the presence of a fourth transmembrane domain in 5-HT₃ serotonin-gated channels. In this region, the significant homology between members of this class of neurotransmitter-gated channels suggests strongly that they have a common transmembrane organization basically different from glutamate-gated and ATP-gated channels.     

The reproductive biology of the cyprinid, Thynnichthys thynnoides (Bleeker), in the Chenderoh Reservoir — a small tropical reservoir in Malaysia

The reproductive biology and breeding cycle of a tropical cyprinid, Thynnichthys thynnoides (Bleeker), from Chenderoh Reservoir, Malaysia was studied from June 1991 to November 1992. A total of 329 and 246 mature females and males and 167 immature females were sampled during the study. Three breeding cycles were observed and the cycles coincided with high reservoir water level which resulted in the floodings of the littoral zone. Five and four gonad developmental stages were observed for females and males, respectively. Oocyte diameter distribution study indicated the species was a total spawner although clutches of ripe oocytes might be released over an extended time period. Fecundity was related to body weight and both absolute and relative fecundity increased with body size. Absolute fecundity ranged from 26962 ± 1484 to 173520 ± 127420 eggs fish^–1, whereas relative fecundity ranged from 121 ± 53 to 451 ± 259 eggs g^–1 body weight, respectively.     

Rhythmic melatonin secretion in different teleost species: an in vitro study

The rhythmic production of melatonin is governed by intrapineal oscillators in all fish species so far investigated except the rainbow trout. To determine whether the latter represents an exception among fish, we measured in vitro melatonin secretion in pineal organs of nine wild freshwater and six marine teleost species cultured at constant temperature and under different photic conditions. The results demonstrate that pineal organs of all species maintain a rhythmic secretion of melatonin under light:dark cycles and complete darkness, and strongly suggest that most fish possess endogenous intrapineal oscillators driving the rhythm of melatonin production, with the exception of the rainbow trout.Abbreviations LD light:dark - DD dark:dark - NAT N-acetyltransferase - RIA radioimmunoassay     

Screening Actinomycetes for Extracellular Peroxidase Activity

A diverse collection of actinomycete strains were screened for production of extracellular peroxidase activity by adapting a chemiluminescence analysis system developed for horseradish peroxidase-based enzyme-linked immunosorbent assay. Extracellular peroxidase activity was found to be common but quantitatively variable, and this rapid and sensitive screening system permitted identification of a small group of high-producing strains. A range of spectrophotometric assays were compared for the measurement of peroxidase activity in concentrated culture supernatants of two selected thermophilic streptomycetes. Of these, the peroxide-dependent oxidation of 2,4-dichlorophenol was identified as the most robust and reproducible assay for quantitative studies.     

Characterization and co-culture of novel nontransformed cell lines derived from rat endometrial epithelium and stroma

Summary Normal and neoplastic growth of epithelial cells depends on mutual interactions between epithelial and stromal cells. As a tool for the study of the underlying molecular mechanisms, we have developed temperature-sensitive, nontransformed cell lines derived from rat uterine epithelium and stroma by transfecting primary cultures with a temperature-sensitive mutant of the SV40 large T antigen. The epithelial and stromal cell lines obtained shared relevant morphological characteristics with the primary cells from which they were derived. Immunocytochemical analysis showed that the epithelial cell lines expressed the intermediate filament cytokeratin, whereas the stromal lines expressed the intermediate filament vimentin. Alkaline phosphatase activity was present in all cell lines examined. All cell lines were anchorage dependent and did not form foci. One epithelial cell line expressed oxytocin mRNA, a gene product recently shown to be highly expressed in vivo in the uterine epithelium at term. If grown on Matrigel, this cell line formed domelike structures, a further characteristic of its differentiated phenotype. In an attempt to reconstitute an endometrium in vitro, epithelial cells were seeded on top of a layer of stromal cells. Paraffin cross sections showed that this in vitro system consisted of a bilayer structure. Four to five cuboidal epithelial cells were typically anchored atop one stromal cell, forming an endometriumlike tissue. The present in vitro system should provide a useful model for further studies on endometrial functions and epithelial/stromal cell interactions at a molecular level.     

An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region.

Translation of the human hepatitis C virus (HCV) RNA genome occurs by a mechanism known as "internal ribosome entry." This unusual strategy of translation is employed by naturally uncapped picornaviral genomic RNAs and several cellular mRNAs. A common feature of these RNAs is a relatively long 5' noncoding region (NCR) that folds into a complex secondary structure harboring an internal ribosome entry site (IRES). Evidence derived from the use of dicistronic expression systems, combined with an extensive mutational analysis, demonstrated the presence of an IRES within the HCV 5'NCR. The results of our continued mutational analysis to map the critical structural elements of the HCV IRES has led to the identification of a pseudoknot structure upstream of the initiator AUG. The evidence presented in this study is based upon the mutational analysis of the putative pseudoknot structure. This is further substantiated by biochemical and enzymatic probing of the wild-type and mutant 5'NCR. Further, the thermodynamic calculations, based upon a modified RNAKNOT program, are consistent with the presence of a pseudoknot structure located upstream of the initiator AUG. Maintenance of this structural element is critical for internal initiation of translation. The pseudoknot structure in the 5'NCR represents a highly conserved feature of all HCV subtypes and members of the pestivirus family, including hog cholera virus and bovine viral diarrhea virus.     

“Wall-papering” and elaborate nest architecture in the ponerine antHarpegnathos saltator

Summary Excavation of 18 nests ofHarpegnathos saltator from southern India revealed an unusually complex architecture for a ponerine ant. The inhabited chambers are not deep in the ground. The uppermost chamber is protected by a thick vaulted roof, on the outside of which is an intervening space serving as isolation from the surrounding soil. In large colonies, the vaulted roof is extended into a shell which encloses several superimposed chambers. Little openings, which may be encircled by moulded flanges, occur in the upper region of the shell. The inside of the chambers is partly or completely lined with strips of empty cocoons. A refuse chamber is always found deeper than the inhabited chambers; live dipteran larvae (family Milichiidae) are typically present. These elaborate nests represent a large energetic investment, and we speculate therefore that nest emigration is unlikely in this species. Consequently, colony fission may never occur, unlike other ants where gamergates reproduce.