Absence of ganglioside GM1 in astroglial cells from newborn rat brain

An immunohistochemical method utilizing anti-ganglioside GM1 antiserum combined with the peroxidase-antiperoxidase technique was applied to a mixed cell population in primary cultures of newborn rat brain. Ganglioside GM1 was demonstrated to be present in neurons and oligodendroglia, but was absent in astroglia. This demonstration was confirmed using a newly developed biotinylated choleragen-avidin-peroxidase procedure. Primary cultures from newborn rat brain cells that had been subjected to a single treatment with trypsin (first passage) and then cultured for 14 days were predominately (95%) composed of astrocytes that stained positively for glial fibrillary acidic protein but were negative for GM1 ganglioside. This preparation contained only 0.34 nmol ganglioside NeuNAc per mg protein compared to 23.9 nmol gangliosidic NeuNAc/mg protein for a five day culture of newborn rat brain mixed cell culture that had not been subjected to passage. Prolongation of culture time from 5 to 21 days in the latter preparation reduced the ganglioside NeuNAc content to 4.9 nmol gangliosidic NeuNAc/mg protein as the proportion of astrocytes in the culture increased. Ganglioside GM1 could not be detected by TLC analysis of the lipid extract obtained from the “pure” astrocyte culture, although small amounts of GM3 and some polysialogangliosides were detected. About half of the label incorporated upon 24 h incubation of astrocytes in the presence of $\text{N-[}{}^3\text{H]acetylmannosammine}$ appeared in ganglioside GM3. It is concluded that astrocytes in mixed cell primary cultures from newborn rat brain, as well as astrocytes in astroglial preparations derived from such cultures, do not contain ganglioside GM1.     

Inhibition of human erythrocyte Ca++-transport ATPase by phenothiazines and butyrophenones

Indirect immunofluorescent staining revealed that Prostatic Binding Protein, the major androgen-dependent protein in rat ventral prostate $\text{in}\text{vivo}$, is associated specifically with the epithelial cells in primary cell cultures derived from rat ventral prostate. The epithelial cells release Prostatic Binding Protein into the medium during primary culture. $\text{De}\text{novo}$ synthesis of Prostatic Binding Protein is demonstrable during early phases of cell culture. Prostatic Binding Protein is an excellent marker for the identification of functional prostate epithelial cells and for the study of regulation at the cellular level of the synthesis and secretion of a major androgen-dependent prostate protein.     

Deoxyribose 5-phosphate aldolase: an enzyme that peaks in the G2 phase of rat hepatoma cells.

The activity of deoxyribose 5-P aldolase (2-deoxy-d-ribose 5-phosphate acetaldehyde lyase, EC 4.1.2.4) increases three- to fourfold in cultures of rat hepatoma cells that have reached stationary growth and have stopped in the G₂ phase of the cell cycle. We have shown that in cell cultures synchronized by three independent methods the enzyme activity peaks in late G₂, followed by a rapid decline during mitosis. Two compounds, dibutyryl cAMP and isoproterenol, that appear to block these cells in G₂, also cause a two- to fourfold increase in deoxyribose 5-P aldolase activity. It is suggested that this enzyme may play a role in lowering the pools of deoxyribonucleotides in the late $\text{G}{}_2\text{M}$ phase of the cell cycle.     

Glutatmine synthetase activity in the chick brain during postnatal growth. Effect of MSO

Glutamine synthetase activity was estimated in the chick cerebral hemispheres, optic lobes and cerebellum between the 1st and the 30th day of postnatal growth. Glutamine synthetase activity is higher in the cerebellum than in the cerebral hemispheres and lowest in the optic lobes at 1 day after hatching; at 30 days after hatching, it is the same in the optic lobes and in the cerebellum and lowest in the cerebral hemispheres. The great increase of glutamine synthetase activity between the 1st and the 4th day after hatching corresponds to the appearance of the heterogeneity of the chick brain glutamate metabolism. The glutamine synthetase activity is inhibited by MSO $\text{in vivo}$ at a concentration of 100 mg kg ^?1 at values of 87, 90 and 89 % in cerebral hemispheres, optic lobes and cerebellum of 1, 2 and 4-day-old chicks. The enzyme inhibition is less pronounced $\text{in vitro}$ and reaches values of about 25 and 75 % for 1 and 10 mM MSO concentrations respectively in the three brain areas of the 1 to 4-day-old chick and values slightly lower in the 30-day-old chick brain.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Butyric acid: A small fatty acid with diverse biological functions

Butyric acid, a 4-carbon fatty acid, affects morphology, growth rate and gene expression in mammalian cells in culture. Sodium butyrate (0.5 to 3 mM) produces reversible growth inhibition in several mammalian tumor cells in culture, but it causes cell death only in human neuroblastomas and human glioma cells in culture. Sodium butyrate in combination with currently used tumor therapeutic agents produced a synergistic, an additive or no effect on growth of mouse neuroblastoma cells and rat glioma cells in culture. At least in NB cells, the cell death and growth inhibition may be related to the reduction in anaerobic glycolysis. Sodium butyrate increases the expression of one or more differentiated functions in mouse NB cells, mouse erythroleukemic cells, human epidermoid carcinoma, human colon carcinoma cells and Chinese hamster ovary cells. The induction of differentiation by butyrate may in part be related to an increase in the cellular cyclic AMP level. Sodium butyrate increases the activities of several enzymes, whereas, it decreases the activities of some. The increase of some enzymes appears to be correlated to hyperacetylation of histones. $\text{In}$$\text{vitro}$ studies suggest that sodium butyrate may be useful in the management of neoplasms by causing selective cell death, and/or cell differentiation and by increasing the cell killing effect in conjuction with currently used tumor therapeutic agents. Sodium butyrate can also be used as a tool to study the regulation of gene expression in mammalian cells.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Forskolin induction of S-100 protein in glioma and hybrid cells

The S-100 protein level in mouse neuroblastoma (N18TG-2 and NIE-115), rat glioma (C6, C6BU-1, and C6V-1), and hybrid (NG108-15, 140-3, 141-B, NBr10A, NBr20A, NCB20, and NX3IT) cells was determined with a sensitive enzyme immunoassay system that uses a rabbit antibody to bovine brain S-100 protein. S-100 protein was detected in glioma but not in neuroblastoma cells. All seven hybrid cells derived from neuroblastoma and glioma or other types of cells were found to possess a very little or undetectable S-100 protein. The induction of S-100 protein level in prestationary phase cultures of glioma C6BU-1 cells was examined by forskolin, which was a highly specific activator of adenylate cyclase of the cells and produced morphological differentiation. After incubation with 10 microM forskolin for 48 hr, the S-100 protein level increased 2-2.5-fold in C6BU-1 glioma cells whose mean control level was 60 +/- 26 ng/mg protein (+/- SD). The forskolin induction of S-100 protein in the cells was dose dependent, and the concentration of forskolin required for 50% activation of S-100 protein was about 0.6 microM. The increase by forskolin was initiated from 10-15 hr after incubation with it and was inhibited with cycloheximide and actinomycin D. In NG108-15 hybrid cells the induction of S-100 protein was also observed by forskolin as well as prostaglandin (PG) E1 plus theophylline which are known to activate adenylate cyclase of the cells. The results indicate that S-100 protein biosynthesis is genetically controlled in these clonal cells, and that S-100 protein can be regulated in a cAMP-dependent fashion in prestationary cultures.     

Myelin basic protein: a substance that releases immunoreactive growth hormone in vitro.

A protein has been isolated from ovine hypothalamus on the basis of its ability to stimulate release of growth hormone by $\text{in}$$\text{vitro}$ cultures of dispersed pituitary cells. This protein has been identified as being myelin basic protein. With no similar biological activity $\text{in}$$\text{vivo}$, myelin basic protein is thus to be recognized as a potentially interfering substance in any search for the physiological growth hormone releasing factor using $\text{in}$$\text{vitro}$ assay systems.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections

Summary Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehydefixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Insulin receptors in rat brain cortex. Kinetic evidence for a receptor subtype in the central nervous system

Binding kinetics of porcine ¹²⁵I-insulin were studied in synaptosomal and microsomal fractions of rat brain cortex. Receptor binding was temperature- and pH-dependent with optimum at 4°C and pH 8.0–8.3. At 15°C, steady state binding was heterogenous, and Scatchard analysis revealed two classes of receptors with K_d of 2 nmol/l and 40 nmol/l in amounts of 50 pmol/g and 200 pmol/g of membrane protein. Dissociation kinetics were biexponential with $\text{T}\text{1}\text{2}$ of about 5 min and 180 min, and in contrast to other cell-types, not influenced by negative cooperativity. No receptor-mediated insulin degradation was detectable at 37°C in the presence of bacitracin. Insulin analogues inhibited ¹²⁵I-insulin binding with potencies relative to porcine insulin (%): human insulin 100, rat insulin (I+II) 71, coypu insulin 47, rat multiplication stimulating activity 8, porcine proinsulin 5, among which the three last values were significantly higher than in rat liver and fat cells. No competition was observed with porcine relaxin and mouse nerve growth factor up to about 1 μmol/l. Receptors were present in all regions of central nervous system with highest concentrations in the cerebral cortex, cerebellum and olfactory bulb, and lowest in the pons, medulla oblongata and spinal cord. In conclusion, insulin receptors in rat brain cortex are functionally different from other tissues regarding the insulin specificity and the absence of negative cooperativity. It is suggested that an insulin receptor subtype in rat brain mediates the growth activity of insulin on nerve cells.     

Rapid inhibition by cycloheximide of rat hepatic nuclear free and engaged poly(A) polymerase activities

This paper reports the effect of cycloheximide on the activity of rat hepatic nuclear poly(A) polymerase. Three hours after cycloheximide treatment (3 mg/100 g body weight), total rat hepatic nuclear poly (A) polymerase activity was decreased to 50% of the normal level. This conclusion was reached when the enzyme activity was measured either in the whole nuclei $\text{in vitro}$ or with partly purified enzyme preparations. When examined at different times after a single injection of cycloheximide, it was observed that poly(A) polymerase activity decayed biphasically with an initial rapid decay phase reaching a minimum at one hour $\text{(}\text{t}\text{1}\text{2}\text{= 0.8}\text{hrs}\text{)}$, followed by a stable phase thereafter. These results have been interpreted to mean that poly(A) polymerase consists of either a mixture of two structurally distinct populations of enzymes with a different turnover rate, or of a single type of enzyme with a protein factor which is rapidly turning over and which is required for maximal enzyme activity.     

Processing of synthetic somatostatin-28 and a related endogenous rat hypothalamic somatostatin-like molecule by hypothalamic enzymes

Synthetic somatostatin-28 (S-28) as well as a related endogenous rat hypothalamic somatostatin-like compound (3K SLI) were incubated with hypothalamic extracts from which endogenous somatostatin-like immunoreactivity (SLI) had been removed by immunoabsorption. The reaction products were analyzed by gel chromatography, HPLC as well as two different radioimmunoassays for tetradecapeptide somatostatin (S-14) in which S-28 crossreacted either 100% (RIA R149) or < 0.001% (RIA S39). The results indicate that incubation of S-28 with SLI free hypothalamic extracts results in a rapid decrease of total immunoreactivity measured with RIA R149 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 14}\text{min}$). By contrast, with RIA S39 a rise from zero to a peak value at 8 min was measured suggesting the formation of S-14. This was confirmed by subsequent analysis by gel chromatography and HPLC. Using endogenous 3K SLI a decrease of total R149-immunoreactivity with a similar time course ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 17}\text{min}$) was observed simultaneously with the emergence of material that corresponded to S-14. This converting activity seems to be specific for SLI-containing tissues since similar rates of conversion were observed with extracts from cerebral cortex and cerebellum but not with lung and liver extracts.It is concluded that (1) S-28 is converted to S-14 by hypothalamic enzymes; (2) the processing of 3K SLI is similar, suggesting the two molecules are closely related, if not identical, and (3) the regulation of S-28 to S-14 conversion could represent an important mechanism for controlling the functional activity of somatostatinergic cells.     

Increase in striatal acetylcholine levels in vivo by piribedil a new dopamine receptor stimulant

Piribedil, (1–2″-pyrimidyl)-4-piperonyl piperazine), an agent proposed for the treatment of Parkinson's disease, was found to increase acetylcholine levels in the rat striatum and diencephalon but not in the mesencephalon, cerebellum or hemispheres. The effect was most marked in the striatum (greater than 100%) and long-lasting (at least 8 hours after a single administration of 60 mg/kg i.p.). Striatal choline levels were also increased by piribedil but did not parallel at all times and doses the effect on acetylcholine. Furthermore, choline levels were increased in all brain regions except the hemispheres. Striatal choline acetyltransferase and acetylcholinesterase were not affected by $\text{in vitro}$ or $\text{in vivo}$ treatment with even high doses of piribedil. α-Methyl-p-tyrosine was ineffective in blocking piribedil while pimozide, a blocker of dopamine receptors, completely antagonized the action of piribedil on striatal acetylcholine. It is concluded that piribedil produced the increase in striatal acetylcholine by directly stimulating dopamine receptors.     

Regulation of polyamine biosynthesis in normal and transformed hamster cells in culture

Several aspects of polyamine biosynthesis were compared in low-passage hamster embryo fibroblasts and transformed hamster fibroblasts. Earlier studies had demonstrated a larger and longer-lasting induction of ornithine decarboxylase activity in transformed cells than in hamster embryo fibroblasts. The increases in intracellular polyamine concentrations after serum stimulation were much greater in chemically transformed HE68BP cells than in normal hamster fibroblasts. Treatment of confluent cultures with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, greatly potentiated ornithine decarboxylase induction by fresh medium in HE68BP cells, but not in hamster fibroblasts. A similar synergistic effect was observed when transformed cells, but not normal cells, were treated with the combination of insulin and promoter. HE68BP cells were capable of growth in medium containing serum concentrations as low as 0.5%, whereas only concentrations of 5% or more supported the growth of hamster embryo fibroblasts. Low serum concentrations induced ornithine decarboxylase in HE68BP cells but not in normal cells, and a given serum concentration always produced a greater induction of ornithine decarboxylase in transformed than in normal cells.Another enzyme involved in polyamine synthesis, $\text{S-}\text{adenosyl-}\text{L}\text{-methionine}$ decarboxylase was induced in normal and transformed cells by serum-containing medium or tetradecanoylphorbol acetate, but in contrast to ornithine decarboxylase, no synergistic effect was seen in transformed cells exposed to the combination of fresh medium and the tumor promoter. A macromolecular inhibitor of ornithine decarboxylase was readily detected in hamster fibroblast cultures treated with high concentrations of putrescine, but little or none of this inhibitor was found in HE68BP cultures. In both cell types, however, serum induction of ornithine decarboxylase was inhibited under conditions of excess putrescine.The results demonstrate several differences between normal and transformed hamster cells in the regulation of polyamine synthesis.     

Increased synthesis of L-glycerol 3-phosphate dehydrogenase during in vitro differentiation of reaggregating cerebellar cells

Reaggregating cell cultures of neonatal mouse cerebellar cells express many of the differentiated properties of normal developing cerebellum, including the transition for the embryonic and adult isozymes of l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). In order to determine the mechanism leading to increased levels of adult isozyme, aggregates in culture from 2 to 17 days were labeled with radioactive leucine and the relative rate of enzyme synthesis was measured after purification of the enzyme by affinity chromatography on Blue Sepharose 6B. During the course of in vitro differentiation, the relative rate of synthesis increased 100-fold, such that it represented 0.5% of the total protein synthesized in the cytoplasmic fraction of the cell. In vivo, $\text{BALB}\text{cBy}$ mice have twice the level of enzyme activity in the cerebellum as do $\text{C57BL}\text{6J}$ mice. Reaggregating cell cultures of cerebellar cells from these strains of mice also express a difference in the activity level, but only when the cerebellar cells are taken from mice 4 days of age or less. When the relative rates of synthesis of l-glycerol 3-phosphate dehydrogenase were measured in cultures expressing the strain-dependent difference in activity, these rates were found to be approximately twofold greater in cultures of $\text{BALB}\text{cBy}$ cells. In contrast, estimates of the relative rate of enzyme degradation by the double-isotope labeling technique indicate that neither specific enzyme degradation nor degradation of total protein is different in aggregates from the two strains of mice. The results suggest that the genetic mechanisms controlling the levels of l-glycerol 3-phosphate dehydrogenase in the cerebellum during development are intrinsic to the cells and, with the exception of serum factors, are independent of systemic influences.     

Temporal variation of adenine ribonucleotides during the cell cycle of Chinese hamster fibroblasts in culture

Chinese hamster fibroblasts in monolayer culture (Don-C cell line) were synchronized by selective detachment of metaphase cells after brief treatment with colcemid. Replicate monolayer cultures were harvested at intervals after synchronization and ethanolic extracts were prepared for the determination of adenine ribonucleotides with the luciferin-luciferase assay. The level of ATP increased approx. 145% during the cell cycle, with the most rapid increase occurring during the G1 phase. One hour after synchronization (early G 1 phase), 1.3 nmoles of $\text{ATP}\text{10}{}^6$ cells were observed; a maximum of 3.2 nmoles of $\text{ATP}\text{10}{}^6$ cells was reached at 12 h (G 2 phase). The adenylate energy charge, $\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP}\text{)/(}\text{ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}$ was lowest during the G 1 phase (0.7) and increased to 0.9 during the late S and G 2 phase. A slight decrease of energy charge was observed during the second mitosis.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. II. Responsible factors for nonspecific destruction

Cell-free supernatants of thoracic duct lymphocyte cultures which were stimulated in vitro by horse serum on syngeneic fibroblast monolayers are demonstrated to be cytotoxic on syngeneic embryonic fibroblasts by means of a direct cell count using microtest plates. Experimental supernatants showed up to 100% suppression of fibroblast growth at $\text{1}\text{3}$ dilution and up to 96% suppression at $\text{1}\text{4}$ dilution as compared to the control supernatants. Evidence is presented indicating that lymphocytes cultured on mosaic monolayers, which were comprised of syngeneic and xenogeneic fibroblasts, were reacting both to xenogeneic cells and horse serum in the medium at the cellular level. A hapten-to-carrier type relationship is suggested between xenogeneic antigen and horse serum. Absence of horse serum in the test cultures using these lymphocytes resulted in the abrogation of nonspecific toxic activity of lymphocytes while the specific activity, though diminished, remained. This again indicates the difference in the mechanisms underlying the specific and nonspecific target cell destruction by T cells.     

Immunocytochemical and Biochemical Analysis of Carbonic Anhydrase in Primary Astrocyte Cultures from Rat Brain

Carbonic anhydrase (CA) was studied in primary monolayer cultures from neonatal rat cerebral hemispheres with both immunocytochemical and biochemical techniques. In such cultures, which consist predominantly of astrocytes, immunocytochemical staining for CA using antibody raised against the type II enzyme from rat erythrocytes resulted in positive staining of the flat, glial fibrillary acidic protein-positive, astrocytic monolayer. Smaller, process-bearing, round cells that grew on top of the astrocytes stained intensely for CA. We estimated that these cells represented 1% or less of the total cells in the cultures, and they have been identified by others as oligodendrocytes. The intensity of the staining of astrocytes for CA could be increased to that observed in oligodendrocytes when the astrocytes were made to round up and form processes by treatment with 2',3'-dibutyryl cyclic AMP. Enzymatic assays showed that CA activity of the cultures after 3 weeks of growth was 2.5- to 5-fold less than that found for cerebral homogenates from perfused 3-week-old rat brains. However, both activities were totally inhibited by acetazolamide with an I50 of 10(-8) M, confirming that both rat brain and the astrocyte cultures possess the high-activity type II enzyme. CA-II activity was unaffected by treatment of the cultures with a method reported to remove oligodendrocytes. Thus, the immunocytochemical and biochemical studies reported here demonstrate that astroglial cells in primary cultures from neonatal rat brain contain CA-II.     

Corticotropin releasing factor stimulates adrenocorticotropin and beta-endorphin release from AtT-20 mouse pituitary tumor cells

Corticotropin releasing factor (CRF) was tested for its ability to stimulate ACTH and β-endorphin secretion from clonal $\text{AtT-20}\text{D16-16}$ mouse pituitary tumor cells. Release of both hormones was stimulated 4 to 5-fold over the basal release at nanomolar concentrations of synthetic CRF. CRF analogues stimulated $\text{ACTH}\text{β-endorphin}$ release with the same order of potency in the tumor cells as in primary cultures of anterior pituitary cells. A 90-min exposure to CRF elicited a 29–35% increase in total ACTH and β-endorphin immunoreactivity in tumor cell cultures. Dexamethasone markedly inhibited CRF-stimulated and basal ACTH and β-endorphin release. $\text{AtT-20}\text{D16-16}$ cells may serve as a good model system for studying the biochemistry of CRF receptor-mediated events involved in $\text{ACTH}\text{β-endorphin}$ release and synthesis.