Harvey M. Shein

Multiplication and Cytopathogenicity of Simian Vacuolating Virus 40 in Cultures of Human Tissues.

Summary Multiplication of SV40 virus has been demonstrated in primary cultures of several human tissues. The virus multiplied without cytopathic effect during the first passage in these systems. In subsequent passages in kidney cell cultures, CPE was noted which included nuclear changes, increased cell proliferation and necrosis.

MEDIATION BY β‐ADRENERGIC RECEPTORS OF EFFECT OF NOREPINEPHRINE ON PINEAL SYNTHESIS OF [14C]SEROTONIN AND [14C]MELATONIN

Rat pineal organs maintained in organ culture converted [14C]tryptophan to [14C]serotonin and [14C]melatonin. The synthesis of both indoles was stimulated by the presence of norepinephrine or dibutyryl adenosine 3′,5′‐monophosphate. This effect of norepinephrine could be blocked by the α‐adrenergic blocking drug, propranolol, but was not modified by the a‐adrenergic blocking agent, phenoxybenzamine. Neither blocking agent modified the pineal response to dibutyryl adenosine 3′,5′‐monophosphate. Unlike dibutyryl adenosine 3′,5′‐monophosphate, the naturally occurring adenosine phosphates did not stimulate synthesis of [14C]melatonin in vitro.

Cyclic Adenosine Monophosphate: Stimulation of Melatonin and Serotonin Synthesis in Cultured Rat Pineals

Dibutyryl cyclic adenosine monophosphate, like norepinephrine, stimulates the synthesis of labeled melatonin and serotonin from tryptophan labeled with carbon-14 by rat pineals in organ culture. Unlike norepinephrine, dibutyryl cyclic adenosine monophosphate does not enhance the accumulation of labeled tryptophan or protein within the pineal. These findings are compatible with the hypothesis that cyclic adenosine monophosphate mediates some, but not all, of the effects of norepinephrine.

Stimulation of [14C] tryptophan 5-hydroxylation by norepinephrine and dibutyryl adenosine 3′, 5′ monophosphate in rat pineal organ cultures

Abstract Both norepinephrine and dibutyryl adenosine 3′, 5′ monophosphate (DAMP) accelerate the synthesis of [14C] serotonin from [14C] tryptophan by cultured rat pineals. When pineals are incubated with [14C] 5-hydroxytryptophan instead of [14C] tryptophan, neither norepinephrine nor DAMP influences the rate of [14C] serotonin synthesis. These data indicate that one locus at which norepinephrine and DAMP influence the biosynthesis of pineal indoles is the enzymatic hydroxylation of [14C] tryptophan. Since the enzymes which catalyze serotonin biosynthesis in brain and pineal appear to be similar, our data raise the possibility that noradrenergic neurons in brain which make functional synaptic contact with serotoninergic neurons might stimulate serotonin synthesis in these neurons.

Neoplastic Transformation of Hamster Astrocytes in vitro by Simian Virus 40 and Polyoma Virus

Astrocytes in cultures of brain cells from fetal or newborn hamsters undergo neoplastic transformation after infection with simian virus 40 or polyoma virus. Subcutaneous or intracerebral inoculation of the transformed brain cells into newborn or adult hamsters produces progressively enlarging astrocytomas at the sites of injection. Astrocytomas produced by polyomatransformed cell lines are histologically better differentiated, but grow more rapidly and metastasize more frequently, than astrocytomas produced by cell lines transformed by simian virus 40. These observations make available in vitro models of virus-induced oncogenesis in astrocytes and provide simple techniques for obtaining astrocytoma cell lines suitable for screening studies of chemical agents effective against astrocytomas.

Sodium-potassium adenosine triphosphatase activity in N-nitrosomethylurea-induced rat astrocytoma cells

Abstract Sodium-potassium stimulated ATPase activity has been determined on microsamples of experimental astrocytomas produced by the subcutaneous growth in hamsters of cloned N-nitrosomethylurea-induced adult rat astrocytoma cells. ATPase activity in the astrocytomas was 15–18% greater in the presence of optimal concentrations of Na + , K + , and Mg 2+ than when Mg 2+ was the only cation present. Astrocyte NaK ATPase activity was modest compared with that of rat cerebral cortex where neurons and their processes and synapses predominate, but similar in magnitude to that of many normal tissues outside the central nervous system which are able to maintain high K + and low Na + concentrations intracellularly. These results are compatible with other chemical as well as neurophysiological data on astrocytes and suggest that normal astrocytes possess a metabolic mechanism for intracellular regulation of monovalent cations and are unlikely, therefore, to be high sodium cells or to act as a functional supplement to the extracellular space for sodium in brain.

Phospholipid metabolism in cultured neuroblastoma and glioma cells incubated with carbamylcholine and norepinephrine.

Cultures of cloned neuroblastoma cells (N1E) in stationary phase and cloned glioma cells (C21) in confluency showed substantial differences in phospholipid composition. As a percentage of lipid P, N1E contained more phosphatidylcholine, less ethanolamine phosphoglycerides and much less sphingomyelin than C21. When incubated with 32Pi both cell lines incorporated comparable amounts of radioactivity into total phospholipids. In NIE, phosphatidylcholine contained much more and phosphatidylinositol and phosphatidic acid somewhat less label as compared to C21. The presence in the incubation medium of either norepinephrine or carbamylcholine failed to elicit stimulation of 32P incorporation into any phospholipid class.

Biochemical structural components of cloned N-nitrosomethylurea-induced astrocytoma cells grown subcutaneously.

A KNOWLEDGE of the biochemical composition of astrocytes is of significance in understanding their neurobiologic role in relation to neurons, their contributions to the blood-brain barrier, and their reaction to disease processes. Attempts have been made to study the biochemistry of relatively normal astroglial cells by using as tissue sources pathological areas of astrogliosis in the central nervous system, cultured astroglia, and astroglia isolated from brain homogenates. The direct isolation of astrocytes from brain in relatively pure form and in quantities sufficient for chemical analysis has been hindred by the cellular heterogeneity and complexity of membranous cell processes in the central nervous system. Recently, however, normal astroglia have been isolated from immature brain by in vitro cultivation and subcutaneous growth as nodules 3 to 20 mg. in wet weight.1 Progress has also been made in obtaining a purified astroglial fraction (68 to 179 mg. of dry weight of astroglia from 84 brains) by density-gradient centrifugation of rat brain (10 to 30 days of age) softened by treatment with trypsin.3 A more abundant source of astroglial tissue, however, is represented by astrocytic tumors. Since well-differentiated neoplasms manifest many of the biochemical properties of the original cell type, such material, if critically studied, may provide clues to the properties of normal astrocytes. Analysis of human astro-

Adenylate cyclase and phosphodiesterase activity of normal and SV40 virus-transformed hamster astrocytes in cell culture

Abstract Adenylate cyclase and phosphodiesterase activity were determined in pure cultures of astrocytes derived from newborn hamster brains. The activities of both enzymes were of the same order of magnitude as that found in whole cerebrum of newborn hamsters. Phosphodiesterase activity of SV 40 virus-transformed newborn hamster astrocytes was similar to that of normal newborn hamster astrocytes. Adenylate cyclase activity of the virally transformed cells was less than one-half that of the normal astrocytes. The results show that normal and neoplastic astrocytes contain the enzymatic machinery required to synthesize and hydrolyze adenosine 3′, 5′-monophosphate (cyclic 3′,5′-AMP). The findings suggest a role for cyclic 3′,5′-AMP in astrocyte function and indicate a possible mechanism for communication of neurons with astrocytes.

LIPID COMPOSITION AND METABOLISM OF CULTURED HAMSTER BRAIN ASTROCYTES

Abstract— The lipid composition and metabolism of confluent cultures of cells derived from newborn hamster brain and having morphology characteristic of immature astrocytes or spongioblasts was investigated and compared to that of newborn hamster brain dispersions and cloned glioma cells (C6). The cells displayed stable morphology for at least 30 subcultures; thereafter spontaneous transformation occurred. No appreciable changes were observed in either composition or metabolic characteristics of any major neutral lipid or phospholipid class in successive subcultures or following transformation. The overall lipid composition of the hamster astrocyte cultures closely resembled that of newborn hamster brain, but the phospholipid composition showed substantial differences. The cells contained as a percent of lipid P relatively more ethanolamine plasmalogen, choline plasmalogen and sphingomyelin and somewhat less phosphatidylcholine and phosphatidylethanolamine. The phospholipids of the hamster astrocyte and C6 cells were similar. Of the lipid precursors examined, [U‐14C]glucose was incorporated best into all preparations. C6 glioma cells incorporated both [U‐14C]glucose and [1‐14C]acetate most actively. From 69–88% of 32P incorporated into hamster astrocyte phospholipids was present in choline phosphoglycerides, whereas the corresonding figure for hamster brain dispersions was 53%. The ratio of specific activities of phosphatidylcholine to phosphatidylinositol was substantially higher in the cultured cells than in the brain preparations. The small pool of choline plasmalogen in the hamster astrocytes usually achieved the highest specific activity of any phospholipid. When [U‐14C]glucose and [1‐14C]acetate were precursors, the bulk of label in the astrocytes appeared in choline phosphoglycerides and triacyglycerol. Our results indicate that the hamster astrocyte cell line as grown expresses distinctive features of lipid composition and metabolism which are nearly constant through many generations.