Long-term effects of dexamethasone and nerve growth factor on adrenal medullary cells cultured from young adult rats

Summary Normal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2 % of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.     

A lectin-like receptor on mammalian macrophages

Rat Kupffer cells in vitro strongly bind neuraminidase-treated rat erythrocytes but not untreated erythrocytes. Binding between cells is inhibited by preincubation of macrophages with D-galactose and related sugars, but not with unrelated saccharides. We therefore suggest that cell adherence is mediated by a galactose-specific receptor on the Kupffer cell membrane.     

On the fine structure of the regressing ecdysial glands of Carcinus maenas L. (Crustacea decapoda) parasitized by Sacculina carcini thompson

Summary The ecdysial glands (Y organs) of the crab Carcinus maenas regress in the presence of an external parasite, Sacculina carcini. This regression is more or less severe and may lead to complete autolysis. Three gradual stages in this involutionary process are described. In stage I, the gland cells are nearly normal. Nuclei and cytoplasmic organelles remain unchanged, but large vacuoles begin to appear. Stage II corresponds to more or less drastic nuclear pyknosis and cytoplasmic alterations. Myelin figures are large and numerous. Lysosomes and autophagic vacuoles with phosphatase activity are abundant. However, the general cellular architecture remains preserved. Stage III corresponds to irreversible cytolysis; nuclear envelopes and plasma membranes have disappeared. What remains is an accumulation of cellular debris becoming engulfed by circulating hemocytes. Not all of the gland cells of any given Y organ show the same degree of regression; degeneration is asynchronous.Structures seemingly corresponding to absorptive roots of the parasite are seen. Their lumen is coated with microvilli. The putative direct and indirect influences of the rhizocephalan parasite on its host are discussed. Our results on regressing Y organs of parasitized crabs are compared with those on regressing ecdysial glands of insects.Dedicated to the memory of Sir Francis Knowles, the first investigator to examine the ultrastructure of the Y organ of Carcinus maenas We wish to express our thanks to Professor Berta Scharrer for her critical advice     

Cycloheximide resistance in Chinese hamster cells. II. Induction of Chm resistance in Chinese hamster cells by N-nitrosomethylurea.

Mutant Chinese hamster ovarian (CHO) cells with a resistance to 7-10(-7) and 8-10(-7) M cycloheximide (CHM) were induced at mutation rates of 1.9-5.2-10(-3) and 1.6-1.8-10(-3) respectively after treatment with N-nitrosomethylurea (NMU) at 100 mug/ml. The induced mutation rates differed by two orders of magnitude from the spontaneous rate of mutation to CHM resistance.     

Tagging of Escherichia coli proteins with new cassettes allowing in vivo systematic fluorescent and luminescent detection,and purification from physiological expression levels

We designed cassettes allowing the systematic fusion of fluorescent or luminescent proteins preceded by the calmodulin binding peptide tag to the C–terminus of Escherichia coli proteins. The chromosomal insertion, and thus physiological expression level of these fusions, permits the study of protein localization by fluorescent microscopy and protein quantification, in vivo and dynamically in diverse conditions. Furthermore, the calmodulin binding peptide tag allows standard detection, affinity purification, and co–purification experiments. These cassettes are therefore very valuable for the versatility of experiments they make available for a given strain, from biochemistry to dynamic and in vivo studies.     

Bacteriocin activity of Pseudomonas sp. on enteropathogenic bacteria in an artificial aquatic system

The bacteriocin of Pseudomonas sp. strain R10 was active in vitro against several enteropathogenic bacteria: enterotoxigenic Escherichia coli, Salmonella typhimurium, Salmonella typhi, Shigella flexneri and Shigella sonnei. Pseudomonas sp. R10 was studied for 5 d in an aquatic system in the presence of strains of the enteropathogenic species mentioned. All the target strains were inhibited in the presence of bacteriocinogenic Pseudomonas sp. R10 and the highest antibacterial action was observed on the second day. Similar results were obtained with the partially purified bacteriocin, although the greatest bactericidal action, in all the studied target strains, was observed on the first day and the bacterial recounts were slightly higher overall.     

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.     

Evidence for the presence of complex high-molecular mass N-linked oligosaccharides in intranuclear glycoproteins from HeLa cells.

Nonhistone proteins were extracted in 0.4 M NaCl from membrane-depleted nuclei of HeLa cells grown in the presence or the absence of [5,6-3H]fucose. Control experiments strongly suggest that most extracted proteins were indeed nuclear components. Several proteins, present in the 0.4 M NaCl nuclear extract, with M(r) ranging from 35,000 to 115,000 were identified on Western blots as fucosylated glycoproteins owing to their binding to the fucose-specific lectin, Ulex europeus agglutinin I. Results of experiments involving mild alkaline treatment and peptide N-glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N-linked to the polypeptide backbone. Analysis of the N-glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the high-M(r) oligosaccharides to endo-beta-galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N-acetyllactosamine units. [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fucose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains were observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoproteins.