Establishment of ''normal'' nervous cell lines after transfer of polyoma virus and adenovirus early genes into murine brain cells.

Brain cells from murine embryos were transfected with the polyoma virus large T or the adenovirus 5 EIA gene and, simultaneously, with the phosphotransferase coding NeoR gene. The efficiently transfected cells were selected for their resistance to Geneticin (G418) and their ability to clone at low cell density. Subsequently, most of the selected cells could be sub-cloned and continuously grown for 6-18 months so far. Their doubling time varied between 18 and 72 h. From independent transfections, more than one hundred cell lines were established. They did not exhibit a transformed phenotype, but subsequent transfection with the polyoma middle T gene induced their oncogenic transformation. The maintenance and expression of the transferred genes were verified. Most of the analyzed cell lines retained glial properties. These results suggest that the lines obtained as well as a further extension of this in vitro system should be of interest for the study of nervous cell interactions, differentiation and functions.     

Gas-liquid chromatographic determination of human fecal bile acids

A method for the determination of total bile acids in human feces that is suitable for routine application is described and discussed. Bile acids are extracted from freeze-dried feces with acetic acid and toluene, in the presence of the internal standard 23-nordeoxycholic acid. After saponification of the extract, bile acids and the internal standard are methylated and converted by mild chromic acid oxidation into their ketonic derivatives. The resultant mixture of a few stable compounds can be separated and measured quantitatively by gas-liquid chromatography on a methylsiloxane polymer. A reference bile acid mixture including the internal standard is also taken through the entire procedure with each series of samples. It has been demonstrated that, in spite of the omission of the usual purification steps, the method is specific for bile acids.     

Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles

A method is described for preparing, by filtration on Millipore filters, very thin (about 10 µ) pellicles of packed particles. These pellicles can be embedded in Epon for electron microscopic examination. They are also suitable for cytochemical assays. The method was used with various particulate fractions from rat liver. Its main advantages over the usual centrifugal packing techniques are that it produces heterogeneity solely in the direction perpendicular to the surface of the pellicle and that sections covering the whole depth of the pellicle can be photographed in a single field. It thus answers the essential criterion of random sampling and can be used for accurate quantitative evaluations.     

Interet diagnostique d’une injection intracaverneuse de 20 μg de Prostaglandine E1 dans l’impuissance

Intracavernous injection of 20 μg of prostaglandin E1 (PGE1) was carried out in 130 impotent patients. The erectile response was compared to the results of arteriological investigations including nocturnal penile tumescence and rigidity monitoring (NPTR) in 59 patients. The response of 60 patients positively categorized as exclusively psychogenic or vasculogenic was also compared to the pattern of the response to 80 mg of papaverine observed in a previous study by the same authors. The PGE1 test may not discriminate psychogenic from wholly organic patients since its results are not correlated to those of NPTR. It helps for the screening of vasculogenic impotence. Lack of response or a partly rigid response is consistent with this actiology but is not specific for it. A fully response makes it unlikely. Compared to papaverine, PGE1 induces less non rigid responses in psychogenic patients (15% versus 35% with papaverine) and more fully rigid responses in vasculogenic patients (respectively 12% and 5 %). Consequently the specificity of the PGE1 test is higher but its sensitivity lower than that of papaverine so that there is no clear difference in the effectiveness of the tests. Nevertheless the PGE1 test should be preferred, because it is safer. Prolonged erections occured in only 5 patients, and all ceased spontaneously. However 4 presented severely painful erections.     

Coexpression of two thermosensitive defects in a Chinese hamster cell line : Manifestation of a G1 block associated with a mitosis defect

Asynchronous cultures of ts12, an anchorage-dependent derivative of the thermosensitive Chinese hamster cell line ts111, show a rapid drop in [3H]thymidine incorporation with accumulation of the cells in the G1 and in the G2 phases of the cycle, when shifted from 34.5 to 39.4 degrees C. Shift-up experiments carried out after either isoleucine deprivation or synchronization at 39.4 degrees C, locate the execution point of a ts function in late G1 (2.5-3 h before S). However, stimulation of proliferation of a high density-arrested population allows a fraction of the cells to enter S. In addition to the G1 ts defect, ts12 expresses a slight cytokinesis defect at 39.4 degrees C (8-15% binucleate cells). The results suggest that altered processes are taking place at a post-metaphasic stage during the first hours after the shift-up. When populations are synchronized by a thymidine block and released at 39.4 degrees C, multinucleate cells in addition to binucleate cells are observed. Part of these multinucleate cells result from abnormal karyokinesis without inhibition of cytokinesis. Evidence is presented suggesting that excess thymidine allows the re-expression of the multinucleation phenotype of ts111.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.