Second Messengers in Neuronal Signaling a

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71900/1/j.1749-6632.1993.tb23019.x.pd

Phospholipid labelling by [ 32 P]-orthophosphate and [ 3 H]-myo-inositol in the stimulated goldfish brain in vivo 1

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65519/1/j.1471-4159.1972.tb01470.x.pd

DETERGENT-LIKE ACTION OF TETRAPHENYLBORATE ON PHOSPHOLIPID LABELLING IN GUINEA PIG CORTEX SUBFRACTIONS 1

—The addition of 2 × 10 −4 m sodium tetraphenylborate to particulate preparations of guinea pig cerebral cortex increases labelling of phosphatidic acid from Γ-[ 32 P]-ATP two- to four-fold. The effect ‘was observed in all subcellular fractions studied (nuclear, synaptosomal, mitochondrial and microsomal) and is not blocked by the addition of atropine. Changes in phospholipid labelling similar to those induced by tetraphenylborate can be demonstrated with sodium dodecyl sulphate or sodium desoxycholate. It is suggested that tetraphenylborate stimulates lipid labelling by a detergent-like activation of diglyceride kinase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65419/1/j.1471-4159.1974.tb06881.x.pd

Purification and Characterization of p68/70, Regeneration-Associated Proteins from Goldfish Brain

Two acidic proteins (p68/70) previously shown to be associated with regeneration of the goldfish optic nerve were purified 887-fold from brain homogenates of Carassius auratus. Purification to homogeneity was achieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE-Sephacel, Cu 2+ -charged iminodiacetic acid agarose, and gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5 2. From these values a molecular mass of 128 kDa and a frictional coefficient ratio of 1.6 were calculated. Chromatofocusing on a high-resolution DEAE column resolved the protein doublet into three dimeric species of p68, p68/70, and p70. These results indicate that the proteins are highly elongated and associate as homodimers or as a hetero-dimer. Subcellular localization and membrane extraction experiments indicated p68/70 to be a component of the plasma membrane associated primarily through hydro-phobic interactions. p68/70 demonstrated biphasic behavior in phase partition experiments using Triton 114. Analysis of hydrolytic products indicated p68/70 to be a glyco-protein, containing 11% carbohydrate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65206/1/j.1471-4159.1994.62031182.x.pd

Lithium Enhances Muscarinic Receptor–Stimulated CDP-Diacylglycerol Formation in Inositol-Depleted SK-N-SH Neuroblastoma Cells

The psychotherapeutic action of Li + in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li + on muscarinic receptor–stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo -inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 ± 4 nmol/mg of protein. The addition of carbachol to [ 3 H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [ 3 H]CDP-DAG was completely blocked by the addition of 10 Μ M atropine, was not dependent on the presence of Li + , nor was it affected by co-incubation with myo -inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [ 3 H]CDP-DAG was potentiated ∼ 10-fold by Li + and substantially reduced by coincubation with inositol. The formation of [ 3 H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [ 3 H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol ≥ oxotremorine-M > bethanecol ≥ arecoline > oxotremorine > pilocarpine. Extended culture of the SK-N-SH cells in an inositol-free chemically defined growth medium progressively reduced the intracellular inositol content to <5 nmol/mg of protein, a level comparable with that seen in cortical slices. In these inositol-depleted cells, Li + potentiated carbachol-stimulated [ 3 H]CDP-DAG formation, and this effect was completely reversed by coincubation with inositol (EC 50 0.2 m M ). The present study thus demonstrates, in the same cultured cell line, the effects of normal and reduced intracellular inositol levels on the ability of Li + to attenuate phosphoinositide resynthesis, as inferred from [ 3 H]CDP-DAG accumulation. The results indicate that Li + can lead to a slowing of stimulated phosphoinositide turnover in neuroblastoma cells, provided that the intracellular inositol content has been significantly reduced.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65723/1/j.1471-4159.1993.tb03289.x.pd

FATTY ACIDS OF THE BRAIN IN PHENYLKETONURIA *

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66194/1/j.1471-4159.1964.tb11456.x.pd

RAPID TRANSPORT OF PROTEIN IN THE OPTIC SYSTEM OF THE GOLDFISH 1

Several amino acids, particularly [ 3 H]proline and [ 3 H]asparagine specifically and efficiently labelled rapidly transported proteins in the goldfish optic nerve and tectum after intraocular injection. Studies with these amino acids showed that the rapidly transported proteins moved as a discrete band at a rate which was temperature-dependent, and was equal to 70-100 mm per day at 20°C. Transported protein in the optic tectum was 80 per cent particulate and was found in synaptosomal, mitochondrial, and myelin fractions, but not in purified nuclei or ribosomes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66053/1/j.1471-4159.1971.tb11965.x.pd

Cloning of CDP-Diacylglycerol Synthase from a Human Neuronal Cell Line

A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP-diacylglycerol, catalyzed by CDP-diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP-diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP-diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP-diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65959/1/j.1471-4159.1996.67052200.x.pd

William Hogarth, Unwitting Neurochemist?

William Hogarth's famous etching Gin Lane is often used to illustrate the debilitating results of alcohol addiction. Less well known is the companion etching Beer Street in which death, murder and squalor are replaced by health, orderliness and joy. Some 250 years later, the rise of science, and specifically of neurochemical research, has defined how the malnutrition, including avitaminosis, resulting from addiction to distilled spirits (rather than more judicious use of less potent alcoholic beverages) disturbs brain metabolism and function. These two etchings, which have survived for their historical and artistic value, continue to have sociological and clinical relevance.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45420/1/11064_2004_Article_290427.pd

II. Biochemical mechanisms in the phosphatidylinositol effect

Stimulated labeling of phospholipids from 32Pi is a hallmark of activation of a variety of cell surface receptors. In the case of the central nervous system, the response can reflect muscarinic activation. Recent studies in nerve ending preparations indicate a postsynaptic site of action. Ca2+ is required for the expression of cholinergic stimulation of labeling in nerve ending preparations, but whether it plays a regulatory role is not yet known. While it is inferred that the receptor-ligand interaction leads to increased diacylglycerol availability, its source is not established. In experiments with muscarinic agents and ionophore added to nerve ending preparations, there is a potentiated loss of labeling from prelabeled polyphosphoinositides. It is suggested that phosphodiesteratic cleavage of polyphosphoinositides may be an early consequence of muscarinic receptor activation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25221/1/0000661.pd