Effect of prenatal exposure to alcohol on membrane-bound enzymes during astrocyte development in vivo and in primary culture

In the present work we have analyzed the effect of prenatal ethanol exposure on the activity of several glial marker and functional enzymes during the development of astrocytes isolated from rat brain as well as in primary culture. The activity of marker enzymes glutamine synthetase and butylcholinesterase showed no differences between isolated astrocytes from 15 and 70 day old control rats. However, the activity of the membrane-bound enzymes (Na+K)ATPase and 5'-nucleotidase was higher in astrocytes from 70 day old control rats than in those from 15 day old animals. Although the pattern found in astrocytes from alcohol-exposed rats was similar to that of controls, the levels of activity of the enzymes were lower in alcoholic than in control animals. When control astrocytes in primary culture were used, the activity of (Na+K)ATPase and 5'-nucleotidase increased throughout the entire culture period. In contrast, the maximal activity of glutamine synthetase was found at 7 days of culture. Ethanol also induced a decrease in the activity of all enzymes, which was more evident at the end of the culture period. These results indicate that the activity of the enzyme markers analyzed increased mainly during the first weeks of life and remained constant after this period. By contrast, the membrane-bound enzymes studied showed a progressive increase with age. In conclusion, since these astrocyte enzymes are important in the regulation of several neuronal functions through the control of the composition of extracellular fluid, the effect of ethanol on their activities could explain some of the neuronal alterations reported in children and animals exposed to ethanol during development.     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.     

Proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase in leaves of Citrus explants throughout the annual cycle and its regulation by ethylene

Leaf senescence and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) degradation in orange [ Citrus sinensis (L.) Osbeck cv. Washington Navel] explants have been investigated. Explants consisted of a segment of stem (ca 15 cm) and 5 mature leaves. In vitro RuBP carboxylase degradation was determined by culturing the explants in water for different periods of time (3 days usually) and quantifying the two RuBP carboxylase subunits in the extracts following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vitro RuBP carboxylase degradation was estimated by autodigestion of leaf extracts and SDS-PAGE. The extent of in vivo RuBP carboxylase degradation in explants cultured under 16 h light/8 h dark photoperiod varied throughout the year and showed a cyclic behaviour correlated with the growth cycle of Citrus. The highest proteolytic activity both in vivo and in vitro was found in explants made from April to August coinciding with the maximum vegetative growth period of the tree.
Leaf senescence and abscission could be retarded significantly at any time of the year by maintaining the explants continuously in the dark. Treatment of the explants in the dark with a continuous flow of ethylene enhanced both leaf abscission and rate of RuBP carboxylase degradation, proportionally to ethylene concentration (0.1-0.6 ppm). Ethylene-induced senescence of Citrus leaf explants in the dark appears to be a convenient model system to study the regulation of the proteolytic degradation of RuBP carboxylase.     

Evolution of several cytoskeletal proteins of astrocytes in primary culture: Effect of prenatal alcohol exposure

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein -tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of -tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti - and anti--tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin.In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed in vivo.Special issue dedicated to Dr. Santiago Grisolia     

Nucleic acid synthesis inChenopodium rubrum L. during photoperiodic induction and its relation to endogenous rhythmicity

Beginning with the second inductive cycle the rate of nucleic acid (NA) synthesis in cotyledons and apical buds ofChenopodium rubrum is higher at the end of the dark period or 4h following transfer of the plants to light in induced plants than in non-induced ones. This is due to an increase in all NA fractions. The greatest difference between NA synthesis in induced and non-induced plants was observed at the end of the second (or sometimes third) inductivecycle. In the subsequent cycles the difference decreased or disappeared eventually. During photoperiodic induction NA synthesis shows a diurnal rhythm with a peak at the end of the dark and at the beginning of the light period. Rhythmicity of NA synthesis is endogenous. The period length of the endogenous oscillation is about 18 h. Interruption of the dark period by light causea amplitude of the first oscillation to be reduced and delays the appearance of the second peak. NA synthesis did not show rhythmicity in plants grown in continuous light. The significance of the observed phenomena for photoperiodic induction is being discussed.     

Changes in the level of endogenous gibberellins and auxins in apical buds ofChenopodium rubrum L. after application of growth substances reversing the effect of (2-chlorethyl)-trimethylammonium chloride (CCC) on flowering

The application of CCC at concentrations inhibiting flowering ofChenopodium rubrum reduces the level of endogenous gibberellins in the apical buds of the plants. The effect of CCC may be reversed by appropriate concentrations of gibberellin (GA-), indole acetic acid (IAA) or kinetin. Kinetin applied to the apical bud during floral induction reduced the level of endogenous gibberellins similarly as CCC and if both CCC and kinetin were applied simultaneously their action was additive. On the other hand IAA applied under the same conditions increased the level of endogenous gibberellins and after joint application of CCC and IAA their level was the same as in untreated control plants. After application of CCC during floral induction the level of endogenous auxins did not change markedly but an active substance “x” appeared on the chromatograms of indole compounds. This substance was found also after simultaneous application of GA- and CCC but not after joint application of CCC and kinetin. If follows from our results that the same morphological phenomenon (flowering) can take place in plants considerably differing as to their level of endogenous growth substances. The ratio of different growth substances is obviously more important than the actual level of the single substances.     

A high level of hybrid dysgenesis in Drosophila: High thermosensitivity, dependence on DNA repair, and incomplete cytotype regulation

Summary An unusually high level of P-M hybrid dysgenesis in Drosophila melanogaster is characteristic of hybrid offspring originating from both, A (M × ) and B (P × M) crosses of a subline of the Harwich P strain, termed H ^s . The novel properties induced by mobility of P elements carried by H ^s paternal chromosomes include: very high (over 95%) gonadal dysgenesis (GD) in both sexes at the low restrictive temperature of 21°C, and highly premature sterility when males are reared at 18°C and aged at 21°C. Although all three major chromosomes of the H ^s subline contributed to this atypical pattern of gonadal dysgenesis, chromosome 3 had the largest effect. Gonadal dysgenesis showed a temperature- and sex-dependent repression pattern by the defective P elements of Muller-5 Birmingham chromosomes; at 21°C there was virtually no repression of male sterility, but most effective repression of GD in females. At 29°C repression was effective in males, but declined in females. The high thermosensitive sterility, low fecundity, and premature aging of the male germ line were greatly exacerbated when males derived from either A or B crosses were deficient either in excision repair (mei-9 mutation) or in post-replication repair (mei-41 mutation). These findings demonstrate that both DNA repair pathways are essential for the repair of lesions induced by P element transposition and support the hypothesis that P element-induced chromosome breaks are responsible for the virtual abolition of the germ line. The relatively high premature sterility of cross B DNA repair-deficient males, reared at 18°C and aged at 21°C, indicates that there is incomplete cytotype regulation in H ^s subline hybrids.