P.F. Urban

DEVELOPMENTAL PATTERNS OF GANGLIOSIDES AND OF PHOSPHOLIPIDS IN CHICK RETINA AND BRAIN

Abstract— The changes in phospholipids and gangliosides during ontogenesis of chick retina have been compared with those in brain. Three phases of accumulation of ganglioside NeuNAc in the retina were detected. In contrast, brain NeuNAc rapidly increased during embryonic life until hatching, followed by a slower increase up to the adult stage. The phospholipid changes in retina and in brain occur in a‐similar manner to the variations observed for gangliosides, however in retina the changes of phospholipid content are less marked than in brain, during embryonic life. There were marked changes in the retina and brain ganglioside patterns with age. Gd3 and Gd1b decreased rapidly in per cent; correspondingly, Gd1a increased during embryonic life and became the major ganglioside in place of Gd3. There was a similarity between ganglioside patterns of chick retina and brain. Except for some slight variations during embryonic life, the retinal phospholipid pattern did not change noticeably.

Direct thin-layer chromatography of gangliosides of a total lipid extract.

A new method of thin-layer chromatography of gangliosides has been developed. The method does not need the preliminary purification of the gangliosides so that a total lipid extract can be applied to a precoated silica gel plate. Under these conditions, gangliosides are not lost as is the case in other procedures involving ganglioside partition in a water phase, dialysis, hydrolysis, etc. Monodimensional ascending chromatography is performed in a sandwich chamber placed in a tank protected from draughts and temperature variations (24 ± 1°C). Three successive solvent systems are used: (1) chloroform; (2) chloroform-methanol-water (70-30-4, v/v/v); (3) chloroform-methanol-0.25% KCl (60-35-8, v/v/v). The procedure has been used for the ganglioside distributions of chicken brain and retinal gangliosides as well as for different brain areas of an inbred mouse strain. The brain and retinal gangliosides pattern are similar to those obtained with purified gangliosides. GD1a in the major ganglioside and appreciable amounts of GM1, GD3, GD1b, and GT1 have been found. The whole ganglioside pattern of cerebellum, forebrain, hippocampus, pons and medulla and the remaining brain are given. The ganglioside distribution differs among the different brain regions with the predominance of either GD1a or GT1.

EFFECT OF LIGHT ON GANGLIOSIDES FROM CALF RETINA AND PHOTORECEPTORS

—The gangliosides of the whole calf retina and the rod outer segments have been analysed. This has been done in two functional states: before and after stimulation by light. After exposure to light no statistically significant change in the gangliosides of the whole retina was observed, but a 40 per cent increase in concentration was found in the rod outer segments. This difference was apparent only when using the same batch of rod outer segments. The major ganglioside in the whole calf retina is GD3 which accounts for 46 per cent of the total. Three other gangliosides GD1a, GD1b and GT1 are quantitatively important, each being between 12 and 16 per cent. GQ1, GM1, and GM3 are minor constituents. In contrast to the chicken retina, GM2 was not detected. The ganglioside N‐acetylneuraminic acid of the rod outer segments accounts for only 1 per cent of the gangliosides of the whole retina. The composition of the gangliosides in the rod outer segments is essentially the same as that of the whole retina. No difference in the relative proportion of the gangliosides of either the rod outer segments or the whole retina was observed after exposure to light.

Enzymatic synthesis of lactosylceramide by a galactosyltransferase from developing chicken retina

Abstract The activity of UDP-galactose: glucosylceramide galactosyltransferase was studied during chick retina ontogenesis. This galactosyltransferase catalyses the formation of lactosylceramide, a reaction which is considered to be one of the initial steps of ganglioside biosynthesis. The activity was detected in 7 day-old embryos and was present in adult animals. The highest specific activity was found at the 11th day of embryonic life. The radioactive product was identified as lactosylceramide by comparison with standard glycolipids, by radiochromatography, by thin layer chromatography of the o -methyl-glycoside derivatives and by gas-liquid chromatography. The enzyme required the presence of Mn 2+ , had a pH optimum of about 6·7 and was stimulated by Triton X-100.

Gangliosides of whole retina and rod outer segments

Few studies have been done on the gangliosides of both the whole retina and the outer segments of the rod photoreceptor cells. Lowry et al. [1 ] suggested that gangliosides may be present in the rod outer segments (ROS). The presence of gangliosides in the whole retina of the ox and calf has been reported by Kostic et al. [2] and Handa and Burton [3]. Very recently Holm et al. established the structure of the major gangliosides from the whole retina of three mammalian species: human, ox and rabbit [4]. We report here a qualitative and quantitative analysis of the gangliosides from the whole retina of two mammals (calf and rat) and a bird (chicken). We also report an analysis of the gangliosides of the calf ROS. The ganglioside composition of the chicken retina is entirely different from that of the two mammals; the ganglioside distribution of the calf ROS is similar to the whole retina.

Brain and Retinal Ganglioside Composition from Different Species Determined by TLC and HPTLC

Gangliosides are compounds found in the CNS at relatively high concentrations (MORGAN et al., 1971, 1973; TETTAMANTI et al., 1972; BRECKENRIDGE, GOMBOS and MORGAN, 1972). In establishing the physiological role of gangliosides, their distribution in different brain areas, cell types, and subcellular fractions should be useful. The presence of a high amount of gangliosides in plasma membranes and in synaptic junctions has been established (HAMBERGER and SVENNERHOLM, 1971; WIEGANDT, 1967).

The effect of flash illumination on the endogenous cyclic GMP content of isolated frog retinae

Abstract In isolated, intact frog retinae high concentrations of cyclic 3′,5′-guanosine monophosphate were found. These levels decline rapidly upon flash-illumination. A significant drop of the endogenous cyclic 3′,5′-guanosine monophosphate content was obtained only 3 sec after a weak light flash which bleached a percentage of the rhodopsin not detectable by customary spectrophotometric methods. No effect of light on retinal cyclic 3′,5′-adenosine monophosphate was found. Cyclic 3′,5′-guanosine monophosphate levels in dark or light were not influenced by 10 m m -aspartate indicating that the light-effect did occur in the photoreceptor cells.

PHOSPHOLIPID METABOLISM IN LIGHT AND DARK ADAPTED EXCISED RETINA

Abstract— The phospholipid composition of, and the incorporation of labelled phosphorus into the different phospholipids of rat and calf retina have been studied. The influence of various conditions, such as dark and light adaptation, during the preparation of retina, lipid extraction and incubation of retina with radioactive phosphorus was investigated.

ENZYMATIC BIOSYNTHESIS OF ETHANOLAMINE- AND CHOLINE-PHOSPHOGLYCERIDES IN RETINA DURING DEVELOPMENT. EFFECT OF LIGHT STIMULATION

Choline‐ and ethanolamine‐phosphoglycerides (CPG and EPG) are the most abundant phospholipids of retinal membranes. We have investigated some regulatory mechanisms involved in the final steps of their biosynthesis, namely those catalysed by CDP‐choline 1,2 diradyl‐sn‐glycerol choline phosphotransferase (CPT) and CDP‐ethanolamine 1,2 diradyl‐sn‐glycerol ethanolamine phosphotransferase (EPT). We have studied both enzymes in the retina which offers an excellent model for the investigation of the molecular basis of the effect of its physiological stimulus, the light. In chick retina. the specific activity (SA) of EPT reached a maximum at the 18th day of embryonic life and decreased thereafter. In the case of CPT, a similar peak of SA was observed at hatching. The time of maximum SA of EPT and CPT corresponded to the period during which retinal rod outer segments are formed. The apparent Km values of EPT and CPT determined with whole retinal homogenates for CDP‐bases showed different profiles. The apparent Km of EPT decreased during embryonic life and increased thereafter whereas the apparent Km of CPT did not change during ontogenesis. Light stimulation of calf retinal homogenates had different effects on phosphotransferase activities. In the presence of only endogenous diacylglycerol (DAG) the SA of CPT was 2‐fold higher for dark‐adapted retinas, whereas no differences in EPT activities were observed. After addition of exogenous DAG (4mM) to the incubation medium, light stimulation of the retina led to a 50% increase of EPT activity whereas no effect was observed for CPT. These different effects could be related to the cyclic nucleotides present in retina before and after light stimulation. In addition all the data presented in this study indicate that, as in brain, CPT and EPT in retina are two different enzymes.

Gangliosides and phospholipids from frog and duck retina.

Abstract Phospholipids and gangliosides have been analysed in duck and frog retinas. Expressed per g wet weight frog retina is richer in phospholipids and poorer in gangliosides than are duck retina and retinas of other species investigated. The phospholipid and ganglioside patterns are very different in the two species. In frog retina, choline phosphoglycerides and cardiolipin are higher and ethanolamine phosphoglycerides, inositol phosphoglycerides, sphingomyelin and phosphatidic acid much lower than in duck retina. In the latter species, ethanolamine plasmalogen levels are nearly twice as high than in frog retina. In the bird retina there are higher amounts in monosialogangliosides (G M1 and G M3 ), different distributions in disialogangliosides (G D3 , G D1a , G D1b and G D2 ) and a lower contribution of tri- and tetrasialogangliosides (G T1 and G Q1 ) than in frog retina. In duck retina we found a new ganglioside called G Y , characterized by an R F lower than that of G Q1 . In general, from the lipid analysis of retinas of several species, phospholipid pattern is rather stable and the ganglioside distribution more variable.