R. Massarelli

Enzymes of lipid metabolism II

Phospho1ipase AZ from cobra venom (Naja naja naja) is a homogeneous, heat-stah1e enzyme that has a monomer mo1ecu1ar weight of on1y 11,000 and contains one histidine and one tryptophan residue. This enzyme acts optimallyon phospho1ipids contained in mixed mice11es with the nonionic detergent Triton X-100; the interactions of this detergent yith pho1§ho1ipid in the mixed mice11es have been e1ucidated with --Hand C-NMR. In reacting, the enzyme first associates with the mixed mice11e and then exhibits surface dilution kinetics in its reaction with substrate. Re cent studies show that various reagents comp1ete1y inactivate this phospho1ipase AZ resu1ting in the modification of the histidine in on1y one-ha1f of the enzyme molecules. These resu1ts suggest that the histidine residue, which is essential for activity, exhibits ha1fsi te reactivity. These and other experiments are interpreted in terms of a model that suggests that the monomeric enzyme forms an asymmetrie dimer or higher order aggregate at the 1ipid-water interface. The studies which are described on the interaction of the phospholipase AZ with mixed mice11es serve as a general model system for understanding detergent effects on the assay of lipid enzymes.

Functional MRI of the human brain: Predominance of signals from extracerebral veins

This study demonstrates the predominance of extracerebral vascular signals in gradient-echo functional magnetic resonance imaging of motor activity at 1.5 Tesla. The demonstration is based upon a novel experimental approach. Maximum intensity projection images are derived from a large set of contiguous 2D functional MR images, and compared with MR angiograms obtained from the volume covered by the set of functional MR images. The comparison shows that the hyperintensities in the functional MR images cover extensive areas, which can be superimposed with a number of veins in the MR angiograms. These results should trigger a general caution in interpretation of the observations in 1.5 Tesla functional MRI.

Guanyl cyclase in chick embryo brain cell cultures: evidence of neuronal localization.

Abstract— Guanyl cyclase activity was studied in dissociated chick embryo brain cell cultures presenting different ratios of neuronal to glial elements. The cultures containing neurons in substantial numbers always had higher guanyl cyclase activities than those consisting mainly of glial cells. No guanyl cyclase activity could be found in cultures made up of pure glial or meningeal cells. These results provide further evidence for our conclusion based on subcellular fractionation studies (Goridis & Morgan, 1973), that brain guanyl cyclase might be overwhelmingly concentrated in neurons. Guanyl cyclase activity of chick embryo cerebral hemispheres increased sixfold between day 12 and day 16 after fertilization; an increase, though of much smaller magnitude, was also seen in cultured cells of the same age.

Effect of Exogenous Gangliosides on the Morphology and Biochemistry of Cultured Neurons

The role of sialoglycoconjugates in various processes essential for the life and development of the neuron is becoming more frequently apparent. In particular, gangliosides have been involved in a large variety of phenomena ranging from cell to cell recognition and adhesion,1,2 to differentiation 1,3 and from possible receptors for neurotransmitters and toxins1,4 to modulators of the movements of solutes across the nerve membranes.5

Effect of CDP-choline on hypocapnic neurons in culture.

Neuronal cultures from chick embryo cerebral hemispheres were protected against a hypocapnic injury by adding to their growth medium 10‐‐6M CDP‐choline before or after the injury. The protection obtained with CDP‐choline was analyzed by a morphometric analysis and showed that pretreatment of neuronal cultures with CDP‐choline maintained the number of cell aggregates and of primary neuronal processes at control values after hypocapnic shock. Various experiments showed that the intact molecule was responsible for the protective action, since pretreatment with different concentrations of various nucleosides and nucleotides (up to 10‐‐5M), choline, and phosphorylcholine was without protective effect. The addition of CDP‐choline after the hypocapnic injury resulted in a protection of the cultures as shown by morphological observation. Incubation of neurons with radioactive choline showed that hypocapnia increased the incorporation of the label into phospholipids whereas the presence of CDP‐choline reduced it. The de novo synthesis of choline was affected by neither hypocapnia nor CDP‐choline treatment. The results indicate that CDP‐choline may have the capacity to protect neurons under conditions of basic pH and that cellular proliferation may be stimulated by the compound.

Surface Glycosyltransferase Activities During Development of Neuronal Cell Cultures

Abstract: Neurons in culture obtained from dissociated cerebral hemispheres of 8‐day‐old chick embryos showed measurable activities of galactosyl‐, fucosyl‐, and sialyl‐transferases at the external surface of their plasma membrane. Important changes in these activities were observed during cell proliferation and maturation, in particular the surface fucosyltransferase activity, and/or the amount of intraceilular fucosylated acceptors increased during synaptogenesis, between 3 and 5 days in culture (d.i.c.). A sodium dodecyl sulfate radioelectrophoretic analysis of the fucosylated neuronal acceptors labelled with [14C]fucose showed, during synaptogenesis, the high labelling of two protein bands of 116 and 50 × 103 daltons. The fucosylation of glycoconjugates occurred preferentially, in neurons, upon glycoproteins whereas in glial cell cultures glycolipids were more fucosylated. The reasons for such a difference are not yet understood but the results suggest that the surface fucosyltransferase activity and fucosylated proteins in particular may play a role during the synaptogenesis of neurons in culture.

The effect of exogenous gangliosides on neurons in culture: A morphometric analysis.

Cultures of isolated neurons have been treated with a purified preparation of gangliosides (10−5M and 10−9M) added to the cell growth medium at the 3rd day in culture and a morphometric analysis of the cells was performed with an image analyzer after 1 and 4 days of treatment. The number of cells and the area of the cell bodies were increased following the treatment. The results indicate as well the ‘sprouting’ effect of the glycolipids on the number of secondary neuronal processes and an increase in the length of the primary neuntes. The present data and other biochemical evidence (Dreyfus et al., 1984, J. Neurosci. Res.) suggest that the addition of exogenous gangliosides may have a trophic effect on neurons, greatly enhances the number of cell to cell contacts, and, possibly, stimulates cell proliferation and differentiation.

Efflux of choline from neurons and glia in culture

The efflux of radioactive choline from exclusively neuronal or glial cell cultures was dependent upon the concentrations of choline present in the cells and in the incubation medium, suggesting the possible presence of a homoexchange phenomenon between influx and efflux. The ionic dependence of the outward movement of choline from these cells showed that is could be stimulated by high K+ concentrations and by the absence of Ca2+. In glial cells, however, the efflux of choline was increased with a much lower concentration of K+ compared to neurons. The result may suggest that during nerve stimulation the release of K+ from neurons could stimulate, from glia, the efflux of choline which would then be taken up in neurons.

A rat brain cytosolic N-methyltransferase(s) activity converting phosphorylethanolamine into phosphorylcholine

It had been previously speculated upon but never proved that the methylation of phosphorylethanolamine could contribute to the production of choline containing compounds. However, experimental evidence obtained with neuronal cultures was interpreted as showing that the stepwise methylation of phosphobases may be an important route for this biosynthesis. We demonstrate that cytosolic fraction from rat brain possesses a N-methyltransferase activity capable of methylating phosphorylethanolamine and its mono- and dimethyl-derivatives into phosphorylcholine. The level of activity detectable in rat liver cytosol is only 18% of that found in the brain cytosol.

Ectoglycosyltransferase activities at the surface of cultured neurons

Glycosyltransferase activities (ectogalactosyl, ectofucosyl and ectosialyl) were studied at the external surface of exclusively neuronal cultures. An appropriate methodology gave the possibility to eliminate sources of errors due to the hydrolysis of nucleotide sugar substrates or due to cellular uptake of free sugars. Ovomucoid and asialofetuin coupled to Sepharose and Ultrogel beads were used as exogenous substrate to circumvent possible substrates pinocytosis. Ectoglycosyltransferase activities were studied as function of protein concentration, incubation time and amount of bead coupled exogenous acceptors. The data show that these enzymes are present at the external surface of the neuronal membrane; their possible role in cell - cell interactions is suggested.