Journal of Neurochemistry

Daval, Jean‐Luc; Vasconcelos, Anne Pereira; Lartaud, Isabelle

doi: 10.1111/j.1471-4159.1988.tb02965.xpmid: 3339344

The responses to diazepam (DZ) and phenobarbital (PhB) were studied in enriched neuronal primary cultures from rat embryo hemispheres. Cells were grown in chemically defined medium and the drugs were added for 3 days to cultures, at pharmacologically active concentrations. Following exposure to DZ or to PhB, morphological changes, such as less prominent neuronal processes, were observed in neurons. It was also shown that each drug reduced the specific uptake of 2‐deoxy‐D‐glucose by the cells and interfered with protein and RNA metabolism. It was concluded that both DZ and PhB might affect, at least transiently, the normal growth of neurons in culture.

Haas, Richard H.; Thompson, Geoffrey; Morris, Bernard; Conright, Kelly; Andrews, Torre

doi: 10.1111/j.1471-4159.1988.tb02966.xpmid: 3339345

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spec‐trophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of (l‐14C)pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units ± 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxy‐lated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units ± 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units ± 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.

Corda, M. G.; Giorgi, O.; Longoni, B.; Ongini, E.; Barnett, A.; Montaldo, S.; Biggio, G.

doi: 10.1111/j.1471-4159.1988.tb02967.xpmid: 2828543

2‐Oxoquazepam (2oxoquaz) is a novel benzodiazepine which shows preferential affinity for type I benzodiazepine recognition sites. In the present study, we analyzed the effect of γ‐aminobutyric acid (GABA), pentobarbital, and chloride ions on (3H)2oxoquaz and (3H)flunitrazepam ((3H)FNT) binding to membrane preparations from rat and human brain. GABA stimulated (3H)‐2oxoquaz and (3H)FNT binding in a concentration‐dependent manner. The maximal enhancement produced by GABA on (3H)2oxoquaz binding was higher than that produced on (3H)FNT binding in both rat and human tissues. In the rat brain, the effect of GABA on (3H)2oxoquaz was similar throughout different brain areas, whereas the effect on (3H)FNT binding was lower in the cerebral cortex and hippocampus than in the cerebellum. Moreover, both (3H)2oxoquaz and (3H)FNT binding were stimulated by chloride ions and pentobarbital. The results are consistent with the hypothesis that type I benzodiazepine recognition sites are linked functionally to the GABA recognition site and the chloride ionophore.

Chantry, Andrew; Earl, Christopher; Groome, Nigel; Glynn, Paul

doi: 10.1111/j.1471-4159.1988.tb02968.xpmid: 2448422

Rat and guinea pig myelin membranes were incubated at physiological ionic strength with millimolar concentrations of Ca2+/Mg2+ ions (37°C; pH 7.4). After 1‐3 h, electrophoresis of the membranes revealed loss of 50% of 18.2‐ and 14.1‐kilodalton (kDa) forms of myelin basic protein (MBP). Concomitantly, peptides representing 25% of the original membrane‐associated MBP were detected in incubation media. Roughly equal amounts of MBP fragments with molecular masses of 10.0 and 8.4 kDa were found in media from guinea pig myelin incubations. Media from rat myelin experiments contained a major 8.4‐kDa and minor 10.0‐ and 5.9‐kDa MBP peptides. Kinetic studies implied that proteolysis occurred subsequent to MBP dissociation from the membranes. Immunoblotting studies indicated that both the 18.2‐and 14.1‐kDa forms of MBP were cleaved near residue 73 to produce a 10.0‐ and 5.9‐kDa C‐terminal fragment, respectively. Degradation of MBP in myelin membranes was partially inhibited by only 5‐20% using leupeptin (20 μM), N‐ethylmaleimide (10 mM), phenylmethylsulphonyl fluoride (1 mM), and phos‐phoramidon (50 μM) but up to 50% by dithiothreitol (DTT, 10 mM). Only DTT and 1,10‐phenanthroline substantially blocked the formation of the characteristic 10.0‐and 5.9‐kDa C‐terminal fragments. This suggests that MBP, dissociating from myelin membrane preparations, is cleaved near residue 73 by a metalloendoprotease distinct from N‐ethylmaleimide/leupeptin‐sensitive calpains and phosphoramidon‐sensitive endopeptidase 24.11.

Abbruzzese, Alberto

doi: 10.1111/j.1471-4159.1988.tb02969.xpmid: 3339346

Deoxyhypusine hydroxylase catalyzes the formation of hypusine from deoxyhypusine in a precursor form of eukaryotic initiation factor 4D (eIF‐4D). The enzymatic activity was examined in mammalian brain homogenates and the results were consistent with the existence of deoxyhypusine hydroxylase levels comparable to those occurring in other mammalian tissues. Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in cow brain (1.82 units/ mg of protein). In the rat the enzyme was found to be unevenly distributed among various brain regions. The parietal cortex contained the highest specific activity (2.1 units/mg of protein). Rat brain deoxyhypusine hydroxylase was mainly present in the postmicrosomal supernatant (81% of the total activity). The highest specific activity (3 units/mg of protein) was observed in the rat brain during the first few days of life. Thereafter the activity started to decline, and continued to do so for 15 days, remaining throughout the rest of life at levels of less than one‐half that of newborn.

Aono, Sachiko; Kato, Taiji; Tanaka, Ryo; Sato, Hiroshi; Semba, Reiji; Kashiwamata, Shigeo

doi: 10.1111/j.1471-4159.1988.tb02970.xpmid: 2828544

Developmental changes of cyclic nucleotides were studied in the hypoplastic cerebellum of jaundiced Gunn rats over the period of postnatal days 8 to 30. The mitogenic activity of glia maturation factor was also measured at day 15. In jaundiced homozygotes(jj), the amount of cyclic GMP on a protein basis was not significantly different from that in control heterozygotes (j+) at either day 8 or 15, but at day 30 it was reduced to about 19% of the control. On the other hand, a lowered nucleotide level on a wet weight basis in jj rats was already statistically significant at day 15. In contrast to cyclic GMP, the rates of increase of cyclic AMP on a wet weight basis were almost the same in the two groups of rats, but the nucleotide levels on a protein basis at days 15 and 30 were a little, but significantly, higher in jj rats than in j+ rats. The activity of glia maturation factor in jj rats was found to be 1.5‐3 times as high as that in j+ rats. Possible implications of the present results are discussed.

Naito, Shigetaka; Bank, Barry; Alkon, Daniel L.

doi: 10.1111/j.1471-4159.1988.tb02971.xpmid: 3276816

Phosphoproteins in the CNS of the nudibranch mollusc, Hermissenda crassicornis, were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography. After preincubation in artificial sea‐water containing 32P, nervous systems were exposed to elevation of external K+ (100 or 300 mM) for a period (e.g., 30 min) approximating a period of depolarization which occurs during classical conditioning. Elevated external K+ was found to change the state of phosphorylation of three distinct proteins (Mr 56,000, 25,000, and 20,000) in three distinct ways without consistently changing that of any other proteins. Phosphorylation of an Mr 56,000 protein was increased by high K+ about twofold only in the presence of external Ca2+ ((Ca2+)o). Phosphorylation of Mr 25,000 protein, on the other hand, was decreased up to 10‐fold by high K+, irrespective of the level of (Ca2+)o. The effect of depolarization on Mr 25,000 protein phosphorylation most likely represents dephosphorylation rather than proteolysis. This interpretation is consistent with the observations that (a) reappearance of the Mr 25,000 protein occurred in the presence of the protein synthesis inhibitors cycloheximide, puromycin, or anisomycin, and (b) the Hermissenda nervous system apparently contains a NaF and EDTA‐sensitive protein phosphatase capable of de‐phosphorylating Mr 25,000 protein. High K+ also reduced Mr 20,000 protein phosphorylation which was dependent on (Ca2+)o even in normal low K+ (10 mM) medium. Removal of (Ca2+)o enhanced reduction of Mr 20,000 phosphorylation due to the high K+ treatment. Interestingly, reduction of the Mr 25,000 protein phosphorylation was long‐lasting, i.e., its phosphorylation did not fully recover to a control level for at least 30 min after the high K+ conditions had been removed. On the other hand, changes of Mr 56,000 and Mr 20,000 phosphorylation did not persist after depolarization had been terminated. A23187, a Ca2+ ionophore, partially reduced Mr 25,000 phosphorylation. These results suggested that depolarization‐induced modification of protein phosphatase and/or protein kinase activity which regulates Mr 25,000 phosphorylation may be involved in biochemical pathways important for memory storage.

Breen, Kieran C.; Regan, Ciaran M.

doi: 10.1111/j.1471-4159.1988.tb02972.xpmid: 3339347

The postnatal sialylation of individual neural cell adhesion molecule (N‐CAM) polypeptides by a develop‐mentally regulated sialyltransferase in Golgi‐enriched fractions isolated from rat brain is described. The 120‐kilodal‐ton polypeptide of N‐CAM was found to be sialylated at each developmental age examined. This was in contrast to the 140‐ and 180‐kilodalton N‐CAM polypeptides which were only sialylated until postnatal day 10 and from postnatal day 12, respectively. Immunoblotting procedures demonstrated that all N‐CAM polypeptides were expressed in the Golgi fractions at each developmental stage examined. The heavily sialylated “embryonic” form of N‐CAM was found to be reexpressed at postnatal days 10 and 12, a time coincident with extensive fibre outgrowth. The “embryonic” form of N‐CAM incorporated similar amounts of (14C)sialic acid into its constituent polypeptides reflecting the difference in sialic acid to protein ratio, as this form of N‐CAM was virtually undetectable in the immunoblots of postnatal material.

Aono, Sachiko; Sato, Hiroshi; Semba, Reiji; Kashiwamata, Shigeo; Kato, Kanefusa; Eng, Lawrence F.

doi: 10.1111/j.1471-4159.1988.tb02973.xpmid: 3339348

The behavior of marker proteins of glial cells (α‐enolase, β‐S100 protein, and glial fibrillary acidic protein (GFAP)) was investigated quantitatively by using enzyme immunoassay systems during the development of eerebellar hypoplasia in jaundiced Gunn rats. A neuronal marker protein, γ‐enolase, was also measured as a reference. At postnatal day 8 corresponding to the early stage of cerebellar damage, the amount of β‐S100 on a protein basis was significantly higher in jaundiced homozygotes (jj) than in control nonjaundiced heterozygotes (j+), whereas no differences in α‐ and γ‐enolases and GFAP were observed between the two groups of rats. At days 15 and 30, which correspond, respectively, to the advanced and late stages of cerebellar damage, the three glial proteins, especially GFAP, were higher and the neuronal protein was lower in the jj rat cerebellum than in the control. These results are consistent with the reported histological observations that neuronal cells are vulnerable and damaged by bilirubin, whereas glial cells seem to be less sensitive. On the other hand, the amounts of β‐S100 and α‐enolase per cerebellum were significantly lower in jj rats at days 15 and 30, as in the case of γ‐enolase, whereas that of GFAP remained at the same level as the control at day 15 and showed a slight but significant decrease at day 30. The possibility is suggested that β‐S100 and GFAP may be available as biochemical indicators of glial cells, especially in the early and advanced stages of eerebellar damage, respectively, but that a‐enolase is less available.

Smith, Ross; Braun, Peter E.

doi: 10.1111/j.1471-4159.1988.tb02974.xpmid: 2448423

Incubation of bovine CNS myelin with phospho‐lipase C from Bacillus cereus under conditions that lead to extensive phospholipid degradation caused 10% of the myelin protein to be released from the membrane. The myelin basic protein (MBP) was a major component of the dissolved protein. Comparable incubations with phospholi‐pase C from Clostridium perfringens, phosphatidylinositol‐specific phospholipase C from Staphylococcus aureus, or cabbage phospholipase D removed little MBP. However, concentrations of sodium chloride near 1 M and concentrations of divalent metal ions between 50 and 600 mM released typically 9‐12% of the total myelin protein, with MBP again as the predominant component. Repetitive washing with calcium chloride solutions resulted in dissolution of over 90% of the MBP. When myelin was incubated in 1.0 M sodium chloride or 25 mM calcium chloride, the MBP was cleaved largely into two major peptides with apparent molecular weights near 14,000 and 12,000, but with 200 mM or higher concentrations of calcium chloride most of this protein remained intact. With appropriate manipulation of the ionic milieu, it is thus possible to remove most of this extrinsic protein from the myelin surface under relatively mild conditions. The conditions that release the protein suggest that it is held at the membrane surface by ionic interactions.