A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules

With the aim of quickly and easily characterizing new estrogen or anti-estrogen molecules, we developed a cellular model in which estrogenic action can be detected by bioluminescence. This model is based on MCF-7 cells stably transfected with a receptor gene which allows expression of the firefly luciferase enzyme under control of the estrogen regulatory element of the Xenopus vitellogenin A2 gene. A stably transfected cell line (cultured for more than eight months without loss of the chimeric estrogenic response) was established by cotransfection of a neomycin resistance gene and cloning under selective pressure. Subcloning luminescent clones was accomplished by using a single-photon detecting camera. This cellular model allowed the study of an estrogenic activity either in whole-cell or in cell-free experiments by detection of the induced luciferase. Estradiol induced the luciferase activity in a dose-dependent manner at subnanomolar concentrations. The induced luciferase activity reached a maximum level as early as 24 hours after the cells were incubated with estradiol. The antiestrogen 4-hydroxy-tamoxifen inhibited the luciferase activity induced by estradiol. The cross-reactivity of ligands, such as dexamethasone, progesterone, testosterone, aldosterone, calcitriol, oxysterol and retinoic acid, were also studied, showing an estradiol specificity for a 24-hour incubation time.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Effects of alginates and sodium carrageenates mixed with soy proteins on the coefficient of protein efficiency

Male 21 d-old Wistar rats, were fed for 4 wk with diets containing casein or soybean proteins (10%) with 0.5, 1, 2 or 3% sodium alginate or sodium carrageenan or without any alginate or carrageenan. Daily protein intake and weight gain of casein-fed rats were not significantly different (P less than 0.05) from those of rats fed soybean meal with alginate, whatever the dose received. Rats fed 3% carrageenan in soybean meal had significantly higher feed intake than that of rats fed casein. At the levels studied, alginate had no effect on the Protein Efficiency Ratio (PER), but carrageenan did. The addition of increased quantities of carrageenan to soybean meal followed by heating the mixture led to a progressive and significant decrease in PER at all levels of carrageenan compared to casein feeding. The addition of 3% carrageenan to heated soybean meal, corresponding to 0.62% of meal diet, led to a significant decrease in PER. These results confirm the precipitating role of carrageenans on proteins.     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Control of myogenesis in the mouse myogenic C2 cell line by medium composition and by insulin: Characterization of permissive and inducible C2 myoblasts

Using subcloning and manipulations of culture conditions we have isolated from the mouse myogenic cell line C2 a variant cell line that we named inducible. Unlike the progenitor cells that are referred to as permissive, inducible myoblasts differentiate poorly in Dulbecco modified Eagle medium plus fetal calf serum (FCS) and require the presence of insulin at a high concentration (1.6 10(-6) M) or insulin-like growth factor I (IGFI) at a lower concentration (2.5 10(-8) M) to differentiate. Permissive and inducible myoblasts fail to differentiate when grown in MCDB202 medium plus 20% FCS, even after a prolonged arrest in G1 phase. This shows that an arrest in G1 is in itself insufficient to trigger terminal differentiation. Both cell types also exhibit distinct patterns of accumulation of muscle mRNAs corresponding to sarcomeric actins and myosin light chain MLC1A. The possibility that these two cell lines might represent two different stages of the progression of myoblasts toward terminal differentiation is discussed.     

Distribution of glutamate decarboxylase-like immunoreactivity in the sixth abdominal ganglion of the cockroach Periplaneta americana

Summary An antiserum against glutamate decarboxylase (GAD) of the rat brain was used to locate GAD activity in sections of the nervous system of the cockroach, Periplaneta americana. The sixth abdominal ganglion was chosen because electrophysiological evidence suggests the presence of GABAergic inhibitory synapses in the cereal-giant interneuron system. Groups of somata and numerous fibres and tracts were positively labelled by the GAD antiserum. A posterior group of labelled somata could be identified close to the entry of the cereal nerves. A line of somata clusters lay along a ventro-lateral furrow. Another discrete row of GAD-like cells was located dorso-laterally. Some small cells among the dorsal unpaired neurons were labelled. A small central group appeared under these cells. An abundance of GAD-like processes and transversal tracts were found within the neuropile. The different systems of GABAergic inhibitors in the ganglion are discussed; in particular we show that the fibres of cereal nerve X are not labelled. This demonstrates that the latter act on the giant fibres via interneurons. We suggest that the group that sends axons into the overlapping region between the cereal nerve and the giant fibre could be the inhibitory interneurons involved in this system.     

Effects of training at simulated altitude on performance and muscle metabolic capacity in competitive road cyclists

Differences between the effects of training at sea level and at simulated altitude on performance and muscle structural and biochemical properties were investigated in 8 competitive cyclists who trained for 3-4 weeks, 4-5 sessions/week, each session consisting of cycling for 60-90 min continuously and 45-60 min intermittently. Four subjects, the altitude group (AG), trained in a hypobaric chamber (574 torr = 2300 m above sea level), and the other four at sea level (SLG). Before and after training work capacity was tested both at simulated altitude (574 torr) and at sea level, by an incremental cycle ergometer test until exhaustion. Work capacity was expressed as total amount of work performed. Venous blood samples were taken during the tests. Leg muscle biopsies were taken at rest before and after the training period. AG exhibited an increase of 33% in both sea level and altitude performance, while SLG increased 22% at sea level and 14% at altitude. Blood lactate concentration at a given submaximal load at altitude was significantly more reduced by training in AG than SLG. Muscle phosphofructokinase (PFK) activity decreased with training in AG but increased in SLG. All AG subjects showed increases in capillary density. In conclusion, work capacity at altitude was increased more by training at altitude than at sea level. Work capacity at sea level was at least as much improved by altitude as by sea level training. The improved work capacity by training at altitude was paralleled by decreased exercise blood lactate concentration, increased capillarization and decreased glycolytic capacity in leg muscle.     

Expression of the HNK-1/NC-1 epitope in early vertebrate neurogenesis

Summary A family of glycoconjugates has recently been shown to share a common carbohydrate epitope recognized by the mouse monoclonal antibody HNK-1. The specificity of HNK-1 was found to be similar to that of another monoclonal antibody, NC-1. These two IgM monoclonal antibodies were raised after immunization of mice with a human T-cell line and avian neural crest-derived ganglia, respectively. The antigens recognized by these antibodies include the myelin-associated glycoprotein, MAG, a glycolipid of defined structure, and a set of molecules involved in cell adhesion. The timing and pattern of appearance of these antigens are distinct. Moreover, the epitope may be absent on an antigen at a given stage or in a given tissue. Therefore, although the molecules able to carry the NC-1/ HNK-1 epitope are numerous and expressed in various tissues, the use of the monoclonal antibodies on tissue sections has proven adequate for following the migration of avian neural crest cells, the major cell lineage recognized by NC-1 and HNK-1 during early embryogenesis. Analogies in several other species have been found on the basis of HNK-1 reactivity. In this study we show that NC-1 and HNK-1 can be used successfully to label migrating neural crest cells in dog, pig and human. On the other hand, the NC-l/HNK-1 epitope was not present on migrating crest cells in amphibians or mice and was found only transiently on the neural crest of rats.     

Isolation and characterization of a thermophilic Methanobacterium able to use formate,the strain FTF

A thermophilic anaerobic which produced methane from formate and H₂ and CO₂ was isolated from a bench-scale digester treating a mixture of solid wastes at 55°C, after enrichment cultures on sodium acetate. The cells were slightly crooked rods occurring singly or in filaments. The bacterium was not motile, and stained Gram positive. Colonies appearing after 1 week of incubation were white with filamentous edges and 1 mm in diameter. The organism used H₂:CO₂ or formate as an energy source. Yeast extract was not required but stimulated growth significantly. Casamino acids were stimulatory and could serve as a nitrogen source. Cysteine was used as a sulfur source. The optimum pH for growth was 7.5. Growth occurred from 35 to 70°C with an optimum at 55°C. The deoxyribonucleic acid base composition was 49.2 mol% guanine plus cytosine. Though this isolate conforms to Methanobacterium thermoformicium, its proper assignment awaits further studies. It has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 3012.This work was supported in part by the Conseil Régional Nord/Pas-de-Calais