Ultrastructural localization of glucocorticoid receptor (GR) in hypothalamic paraventricular neurons synthesizing corticotropin releasing factor (CRF)

Summary Corticotropin releasing factor (CRF) synthesizing neurons, located in the hypothalamic paraventricular nucleus (PVN), are the main central regulators of the pituitary-adrenal cortex endocrine axis. The hormone production and release of CRF-synthesizing neurons is regulated by neuronal messages and feedback action(s) of glucocorticoids secreted by the adrenal gland. In order to characterize the latter mechanism, glucocorticoid receptor (GR)-immunoreactive (IR) sites were studied in hypothalamic paraventricular neurons of intact, long-term adrenalectomized, and adrenalectomized plus glucocorticoid treated animals, by means of ultrastructural immunocytochemical labelling. In intact animals, glucocorticoid receptor immunoreactivity was found predominantly in the nuclei of parvocellular neurons. Following adrenalectomy GR-immunoreactivity was localized in the cytoplasm of the cells, and there was a concomitant disappearance of the label from the nuclei. After corticosterone administration to adrenalectomized animals, GR-IR sites were again concentrated within the cell nuclei. Immunocytochemical double labelling studies performed on adrenalectomized plus corticosterone-replaced animals demonstrated glucocorticoid receptor-IR sites in the cell nuclei of parvocellular paraventricular neurons that expressed CRF-immunoreactivity in their cytoplasm.These ultrastructural data indicate that the intracellular location of glucocorticoid receptor is dependent on the availability of glucocorticoids by the neurons. The simultaneous expression of GR- and CRF-immunoreactivity in parvocellular paraventricular neurons supports the concept of a direct feedback action of glucocorticoids upon CRF-synthesizing neurons.Supported by NIH Research Grants NS19266 (W.K.P. and Zs.L.), NS20832 (M.C.B.) and a joint grant (INT-8703030) awarded by the National Science Foundation and the Hungarian Academy of Sciences (Zs.L. and W.K.P.). R.M.U. is a recipient of NIMH Pre-doctoral Fellowship and M.C.B. an NIH Research Carcer Development Award     

Inhibition of glycoprotein oligosaccharide processing in vitro and in influenza-virus-infected cells by alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene.

The effects of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene (MMNT) on mannosidases involved in asparagine-linked oligosaccharide processing were investigated. MMNT was found to inhibit the activity of rat liver Golgi alpha-mannosidase I in a concentration-dependent manner (50% inhibition with 0.18 mM-MMNT), whereas rat liver endoplasmic-reticulum alpha-mannosidase appeared to be resistant (less than 5% inhibition at 1 mM-MMNT). Jack-bean alpha-mannosidase was also sensitive to inhibition by MMNT (50% inhibition with 0.32 mM-MMNT). Treatment of influenza-virus-infected chick-embryo cells with 1 mM-MMNT led to a decrease in the formation of complex-type asparagine-linked oligosaccharides and an accumulation of high-mannose-type oligosaccharides with the composition Man8(GlcNAc)2 and Man7(GlcNAc)2 on the viral glycoproteins. The biological activities of influenza-virus haemagglutinin and neuraminidase synthesized in the presence of 1 mM-MMNT remained unchanged, but the virus was less infectious than the control.     

Elevated epidermal growth factor receptor binding in plutonium-induced lung tumors from dogs

The objective of this study is to examine and characterize epidermal growth factor receptor (EGF-R) binding in inhaled plutonium-induced canine lung-tumor tissue and to compare it with that in normal canine lung tissue. Crude membrane preparations from normal and lung-tumor tissue from beagle dogs were examined in a radioreceptor assay, using 125I-labeled epidermal growth factor (EGF) as a ligand. Specific EGF receptor binding was determined in the presence of excess unlabeled EGF. We have examined EGF receptor binding in eight lung-tumor samples obtained from six dogs. Epidermal growth factor receptor binding was significantly greater in lung-tumor samples (31.38%) compared with that in normal lung tissue (3.76%). Scatchard plot analysis from the displacement assay revealed that there was no statistical difference in the binding affinity but significantly higher concentration of EGF-R sites in the lung-tumor tissue (619 fmol/mg) than in normal lung tissue (53 fmol/mg). The increase in EGF-R number in plutonium-induced dog lung tumors does not seem to correlate with increase in the initial lung burden exposure to plutonium. Our results demonstrate that there is a significant increase in EGF-R binding in inhaled plutonium-induced dog lung tumors.     

Effects of antimalarial drugs on phospholipase A and lysophospholipase activities in plasma membrane, mitochondrial, microsomal and cytosolic subcellular fractions of rat liver

Activities of membrane-associated phospholipases A1 and A2, and membrane-associated as well as soluble lysophospholipases were measured in different subcellular fractions of rat liver, using suspensions of stereospecifically labelled radioactive phospholipids as substrates. Plasma membranes and endoplasmic reticulum were shown to contain phospholipase A1 and lysophospholipase activities, both of which could be stimulated by Ca2+, mitochondria Ca2+-dependent phospholipase A2 and cytosol Ca2+-independent lysophospholipase activities. Each of these lipolytic enzymes could be inhibited by antimalarial drugs (chloroquine, mepacrine, primaquine) at concentrations above 1 x 10(-4) M. Inhibition of the alkaline cytosolic lysophospholipase by these drugs was noncompetitive with respect to the substrate, and the inhibitory potency increased, when the pH was raised.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Synthesis of polypeptides by the cervix of the baboon (Papio anubis)

Administration of oestradiol to ovariectomized baboons caused the epithelium of the cervix to differentiate into tall columnar cells that were ciliated or secretory. Administration of progesterone in the presence or absence of oestradiol altered the appearance of the lining epithelium, suggesting a decrease in secretory activity. Fluorographs of media from cultures of tissue from steroid-treated animals reflected changes in polypeptide biosynthesis which correlated with the morphological observations: 6 polypeptides (Mr 88,000-37,000; pI 5.5-6.0) were observed in all treatment groups and, except for relative changes in intensity, these polypeptides were electrophoretically similar to those synthesized by the endometrium. A new group of low molecular weight polypeptides (Mr 23,000-20,000, pI greater than 8.0-5.5) and a basic protein (Mr 160,000) were synthesized and released in the oestradiol-dominated animal. These polypeptides were distinct to the cervical mucosa since they were not observed in the endometrium or oviduct. Progesterone suppressed the synthesis of the low molecular weight acidic polypeptides (Mr 23,000-20,000; pI 6.1-5.5) but maintained the synthesis of the basic polypeptides (Mr 23,000-20,000; pI greater than 8). Treatment with progesterone +/- oestradiol did not appear to induce the synthesis of any new major polypeptides in the cervical epithelium. These results suggest that oestradiol induces the synthesis of a group of cervix-specific polypeptides and progesterone antagonizes the action of oestradiol in the baboon cervix.