Inorganic polyphosphates and phosphohydrolases inHalobacterium salinarium

Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 μmol P_i/g wet biomass by the time it entered the growth retardation phase. The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase. The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days). The polyphosphate content ofH. salinarium cells was close to that of yeasts and eubacteria. The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities ofH. salinarium cells were several times lower than those of the majority of eubacteria. The specific activity of pyrophosphatase, the most active hydrolase ofH. salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period. Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol. The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa. The effective K_m of this enzyme with respect to pyrophosphate was 115 μM.     

Pyridoxal phosphatase: cytochemical localization in GERL and other organelles of rat neurons.

A phosphatase, hydrolyzing pyridoxal-5-phosphate (P5P), a physiologically active component of the vitamin B6 complex and an essential co-enzyme in the synthesis of neurotransmitters, has been localized cytochemically in the perikarya of neurons in the peripheral, autonomic and central nervous systems of the rat. Neurons in dorsal root ganglia, sympathetic ganglia and ventral horn of spinal cord were studied by light and electron microscopy, while Purkinje cells, neurons in the dentate nucleus of the cerebellum, thalamus, and hypothalamus were studied by light microscopy only. Optimal conditions for demonstrating this activity in aldehyde-fixed tissue were determined with dorsal root ganglia. At the optimal pH of 5.0, neurons in these ganglia and in all other neurons studied show pyridoxal-5-phosphatase (P5Pase) activity in GERL. Small neurons in dorsal root ganglia also display enzyme activity in the endoplasmic reticulum (ER); activities in GERL and ER are also appreciably high at neutral pH. Small and large neurons in these ganglia, and neurons of sympathetic ganglia, show variable P5Pase activity in the Golgi apparatus. These localizations differ from the usual sites of both acid phosphatase and alkaline phosphatase activities. The P5Pase activity, demonstrated cytochemically, is a new acid hydrolase activity in GERL.     

Corresponding defects in the dorsal fin and in the male breeding crest ofTriturus alpestris

Summary In a wild population ofTriturus alpestris several adult males with an incomplete breeding crest were found. Among the offspring from this population larvae with defects in the dorsal fin were observed. Neither the pigment cells nor the spinal ganglia appear affected in the deficient trunk region of larvae and adults.This work was supported by the Georg and Antoine Claraz-Schenkung. The author wishes to thank Mr. P. Diethelm for ceding the animals and Miss M. Eich for her technical assistance.     

Histochemical study of isolated neurons in culture from chick embryo spinal ganglia

Summary Dissociated chick embryo spinal ganglia neurons, cultivated without direct contact with glial cells maintain some enzymatic activities, for example: carboxylic esterases, succinic-dehydrogenase (SDH), glutamic-dehydrogenase (GDH), monoamine oxidase (MAO), lactico-dehydrogenase (LDH) and alcoholic-dehydrogenase (ADH) for several days periods.Nerve growth factor (NGF) prolongs the maintenance of the mitochondrial enzymes, carboxylic esterases, LDH and ADH in cultures of isolated neurons. Extract of embryonic spinal cord gives almost similar results as NGF.With the technical assistance of Miss E. Darcel.This work is part of the Doctorat ès-Sciences thesis.     

Pseudomonas aeruginosa acid phosphatase and cholinesterase induced by choline and its metabolic derivatives may contain a similar anionic peripheral site

Summary Different compounds derived from choline, and obtained by demethylation or by oxidation of the primary alcohol group with subsequent N-demethylation, were tested as inducer agents of acid phosphatase and cholinesterase in Ps. aeruginosa. It was found that betaine and dimethylglycine were the most effective inducers of both enzyme activities. These metabolites including choline itself, were not inducers of acid phosphatase and cholinesterase in other Gram-negative bacteria such as: Escherichia coli, Salmonella typhimurium, Shigella flexneri, Enterobacter liquefacciens and Proteus mirabilis. The acid phosphatase activities found in these bacteria were not inhibited in vitro by choline, betaine and phosphorylcholine. From these results it may be concluded that the acid phosphatase activity from Ps. aeruginosa is different from the same activity observed in the other bacteria. In addition, it is also shown that Ps. aeruginosa acid phosphatase and cholinesterase were inhibited by a number of compounds containing a positively charged amino group, with methyl or ethyl groups bound to it. These results seem to confirm that Ps. aeruginosa acid phosphatase and cholinesterase may contain a similar anionic site.     

Fluorescence microscopic observations of catecholamines in cultures of the sympathetic chains

Summary A histochemical technique for the demonstration of catecholamines developed by Falck et al. has been successfully applied to the sympathetic chains of rats and mice maintained in vitro. Catecholamines were localized in the nerve fibers, showing identical green fluorescence as in tissue sections of healthy rats. The cultures 8 days in vitro exhibited positive reaction in a few terminals, whereas sister cultures 1 month in vitro showed strong fluorescence reaction in thicker proximal axons and networks of nerve fibers as well. Reactivity of neuron somas became positive after 1 month of cultivation. Application of reserpine in amount of 0.00025 mg/ml for 2 hours resulted in complete disappearance of fluorescence. Furthermore, cultures of spinal ganglia from fetal rat produced no fluorescence reaction with this technique. Therefore, the reaction is specific for sympathetic nervous tissue and reliable for the differentiation of sympathetic neurons from other types of nerve cells.This work was supported by research grant NBO 3173 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service, and research grant No. 355 from the National Multiple Sclerosis Society, New York.     

Zur Chemodifferenzierung des Rückenmarks und der Spinalganglien der Ratte

Zusammenfassung Alkalische Phosphatase, saure Phosphatase, Glukose-6-Phosphatdehydrogenase und NADH-Diaphorase können erstmalig am 13., unspezifische Esterase am 15. Embryonaltag (ET) im Cytoplasma der Neuroblasten des Vorderhorns und der Spinalganglien nachgewiesen werden. Ein Unterschied zwischen zervikalem und lumbalem Teil des Rückenmarks besteht nicht. Während der weiteren Entwicklung breiten sich die Enzymreaktionen in der grauen Substanz nach dorsal aus. Am Ende der Tragzeit entspricht die Verteilung der Fermente der erwachsener Tiere. — Die Azetylcholinesterase reagiert ab 14. ET bis zur Geburt in den Hintersträngen stark positiv und ab 15. ET gleichzeitig in den Vorderhornzellen und Spinalganglien. Nach der Geburt sind die Perikarya der Vorderhornzellen Azetylcholinesterase-frei, dafür reagiert die Zelloberfläche positiv. — Die lysosomale Lokalisation der sauren Phosphatase in den Vorderhornzellen kann sehr früh (15. ET) nachgewiesen werden. Glukose-6-Phosphatdehydrogenase und NADH reagieren in diesen Zellen während der Embryonalzeit diffus. Ab 18. ET reagiert in der grauen Substanz das Scitenhorn bei Nachweis der Glukose-6-Phosphatdehydrogenase und NADH am kräftigsten. — Das Ependym und die Commissura anterior besitzen vom 13. ET bis zur Geburt eine deutliche positive Reaktion für saure Phosphatase, Glukose-6-Phosphatdehydrogenase und NADH. Gliazellen haben während der Embryonalentwicklung keine nachweisbare Enzymaktivität. Diese tritt erstmalig für Glukose-6-Phospahtdehydrogenase und NADH am 1. Lebenstag auf und steigert sich abhängig vom Fortschreiten der Myelinisation. — Die Neurone im Spinalganglion zeigen unterschiedliche Fermentreaktionen, wahrscheinlich als Ausdruck verschiedener Zellaktivität. — Belastung durch Schwimmen zieht keine Veränderungen der Enzymaktivitäten im Rückenmark und Spinalganglion nach sich.

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Chemodifferentiation of the spinal cord and spinal ganglion of the rat

Summary In the cytoplasm of neuroblasts of ventral horn and spinal ganglia alkaline phosphatase, acid phosphatase, glucose-6-phosphate dehydrogenase and NADH diaphorase can be first demonstrated on the 13th embryonic day and non-specific esterase activity on the 15th embryonic day. There are no differences between the cervical and the lumbal spinal cord. During the further development the enzyme activities in the gray matter extend in a dorsal direction. At the end of pregnancy the distribution of enzymes is like that in adult animals. — The acetylcholinesterase reaction is strongly positiv in the posterior column from the 14th embryonic day to birth, and from the 15th embryonic day onward also in the nerve cells of the ventral horn and spinal ganglia. After birth the pericarya of the ventral horn are devoid of acetylcholinesterase. There is, however, a positive reaction on the surface of the cells. — The lyososomal localization of acid phosphatase can be demonstrated on the 15th embryonic day in the nerve cells of ventral horn. Glucose-6-phosphate dehydrogenase and NADH diaphorase exhibit a diffuse reaction in these cells during embryonic life. From the 18th embryonic day onward the lateral horn of the gray matter shows the highest activities of glucose-6-phosphate dehydrogenase and NADH-diaphorase. — In the ependyma and the anterior commissure acid phosphatase, glucose-6-phosphate dehydrogenase and NADH diaphorase can be visualized from the 13th embryonic day to birth. — In glial cells no enzymes can be demonstrated during embryonic life. On the 1st day after birth glucose-6-phosphate dehydrogenase and NADH diaphorase occur. These enzyme activities then increase depending on the degree of myelination. — In the neurons of spinal ganglia the enzyme reactions show marked differences probably indicating functional differences. — Continuous swimming does not lead to demonstrable enzyme changes in spinal cord and spinal ganglia.

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Stipendiat des Deutschen Akademischen Austauschdienstes.     

Alkaline and acid phosphatases from the extensively halotolerant bacteriumHalomonas elongata

Alkaline and acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2, respectively) ofHalomonas elongata were cytochemically localized on the cell envelope. These enzymes were then isolated and partially purified by sonication, ammonium sulfate precipitation, and column chromatography from cells grown in alanine defined medium at 0.05, 1.37, and 3.4M NaCl. Enzyme assays were conducted at pH 5.0 and 9.0 with varying concentrations of NaCl, KCl, and LiCl in the assay buffer. Results showed higher acid phosphatase activity compared with that of alkaline phosphatase; and all enzyme activities were optimal at NaCl concentrations similar to the medium NaCl concentrations for the cells grown at 1.37 and 3.4M. However, minimum enzyme activities were observed for cells grown at the low salt concentration (0.05M). Although samples showed strong activities at some KCl concentrations, generally the enzyme activities decreased significantly when KCl or LiCl was substituted for NaCl. Polyacrylamide gel electrophoresis followed by histochemical staining for the phosphatases showed only one band for both enzymes for each cell sample grown at the different NaCl concentrations.     

Routine enzymes in the monitoring of type 2 diabetes mellitus

We examined the relationships of glucose and HbA1c levels with the routinely screened serum enzyme activities in type 2 diabetes mellitus, and we designed an in vitro study to evaluate the direct effect of glucose levels on enzyme activities. The study was performed on a consecutive series of outpatients with type 2 diabetes who were followed up at Dicle University Medical Faculty Hospital from May 2009 to May 2010 for the first time. Effects of aspartate transaminase, aminotransferase, gamma‐glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) activities, glucose and HbA1c levels and in vitro glucose (492, 287, 184, 131, 82 mg dl⁻¹, respectively) on enzymes were determined. The patients were categorized on the basis of glucose and HbA1c levels and grouped according to a range of values. In patients with high HbA1c levels (>10.1%), ALP, GGT activities and creatine kinase (CK)‐MB/CK (p = 0.008, 0.026, 0.014) ratio were increased significantly when compared with those in the control group. In patients with high glucose levels (>200 mg dl⁻¹), ALP, GGT activities and CK‐MB/CK ratio (p = 0.003, 0.001, 0.001) were increased significantly when compared with those in the control group. Glucose, which was added to serum in different concentrations in vitro, did not directly affect enzyme activities such as ALP, GGT and CK. We concluded that increased glucose levels could damage the liver and the heart muscle cells. Monitoring of blood glucose levels is a more valuable parameter than monitoring HbA1c in the momentary evaluation of diabetes. Copyright © 2011 John Wiley & Sons, Ltd.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Phosphatase activity in the heterotrophic dinoflagellate Pfiesteria shumwayae

The ELF-97 phosphatase substrate was used to examine phosphatase activity in four strains of the estuarine heterotrophic dinoflagellate, Pfiesteria shumwayae. Acid and alkaline phosphatase activities also were evaluated at different pH values using bulk colorimetric methods. Intracellular phosphatase activity was demonstrated in P. shumwayae cells that were actively feeding on a fish cell line and in food limited cells that had not fed on fish cells for 3 days. All strains, whether actively feeding or food limited showed similar phosphatase activities. P. shumwayae cells feeding on fish cells showed ELF-97 activity near, or surrounding, the food vacuole. Relatively small, spherical ELF-97 deposits were also observed in the cytoplasm and sometimes near the plasma membrane. ELF-97 fluorescence was highly variable among cells, likely reflecting different stages in digestion and related metabolic processes. The location of enzyme activity and supporting colorimetric measurements suggest that, as in other heterotrophic protists, acid phosphatases predominate in P. shumwayae and have a general catabolic function.     

CERAMIDE GLYCOSYLTRANSFERASES IN CULTURED RAT CEREBELLUM: CHANGES WITH AGE, WITH DEMYELINATION, AND WITH INHIBITION OF MYELINATION BY 5-BROMO-2''-DEOXYURIDINE OR EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS SERUM

—UDP-galactose:ceramide galactosyltransferase (CGalT) (E.C. 2.4.1.62) and UDP-glucose:ceramide glucosyltransferase (CGlcT) activities were measured in myelinating cultures of newborn rat cerebellum. Specific activities were measured at various days in vitro and the pattern of activities compared with that reported for in vivo tissue. Cultures demyelinated by incubation with media containing 22% serum from rabbits in which experimental allergic encephalomyelitis (EAE) was induced by injection with whole guinea-pig spinal cord, had 28% of CGalT specific activity and 86% of CGlcT specific activity measured in control cultures. Cultures in which myelination was inhibited by maintenance on media containing 0.15 mm -5-bromo-2′-deoxyuridine (BUdR) had 10% of CGalT specific activity and 118% of CGlcT specific activity of control cultures. Cultures in which myelination was inhibited by maintenance on media containing 2% EAE serum had 12% of the CGalT specific activity of control cultures. The data suggest that in vitro CGalT is predominantly a glial enzyme while CGlcT occurs primarily in neurons, and that the reduced CGalT activity may be involved in the mechanism of myelination inhibition by BUdR and by EAE serum.     

Ion-transporting ATPases and matrix mineralization in cultured osteoblastlike cells

Summary Cultures of osteoblastlike cells obtained from the endosteal surfaces of rabbit long bones formed and mineralized an extracellular matrix when they were supplied daily with medium containing fresh ascorbate. No matrix formed without this supplementation. The matrix mineralized whether or not beta-glycerophosphate, a substrate of alkaline phosphatase, was added to the medium. The ion-transporting ATPase activities of untreated, ascorbate-treated, and ascorbate plus beta-glycerophosphate-treated cells were measured. Ascorbate-treated and ascorbate plus beta-glycerophosphate-treated cells had similar enzyme activities. The activities of the Ca²⁺-ATPase; Ca²⁺,Mg²⁺-ATPase; and alkaline phosphatase in treated cells were elevated over the activities in untreated cells. Na⁺,K⁺-ATPase activity was lower in treated than in untreated cells. HCO₃ ⁻-ATPase activity was not changed by treatment. Alkaline phosphatase activity was 20 times higher in freshly isolated osteoblastlike cells than in cells grown to confluence in primary culture. In addition, subculturing further reduced the activity of this osteoblast-marker enzyme. The activities of the ion-transporting ATPases and alkaline phosphatase in second passage cells were similar to the activities of these enzymes in fresh, noncalcifying tissues. Nevertheless, second passage cells retain the ability to mineralize an extracellular matrix, and their ion-transporting ATPase and alkaline phosphatase activities are altered when the cells mineralize a matrix. This work was supported by Grant NAG-2-108 from the National Aeronautics and Space Administration, Washington, D.C., and Grant 5 PO1 NS15767 from the National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD.     

Glycoside hydrolases of rumen bacteria and protozoa

Sixteen strains of rumen bacteria and 21 protozoal preparations were screened for glycoside hydrolase and phosphatase activity, using 22 nitrophenyl glycoside substrates. The range and level of bacterial enzyme activities were species dependent, although, the glycosidases associated with plant cell wall breakdown were most active in the cellulolytic and hemicellulolytic species. Alkaline phosphatase occurred widely in the organisms examined, but was most active in the twoBacteroides ruminicola strains.A wide range of enzyme activities was also detected in the holotrich and Entodiniomorphid ciliates isolated from the rumen or cultured in vitro. The glycosidases involved in cellulose and hemicellulose breakdown were detected in all of the protozoa examined, and, with the exception ofEntodinium spp., were most active in the Entodiniomorphid protozoa; -l-arabinofuranosidase, an essential hemicellulolytic glycoside hydrolase, was particularly active in this latter group of ciliates.     

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

An in vitro production of bone specific alkaline phosphatase

Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadaveric bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented.     

THE CHARACTERIZATION OF HISTIDINE DECARBOXYLASE AND ITS DISTRIBUTION IN NERVES, GANGLIA AND IN SINGLE NEURONAL CELL BODIES FROM THE CNS OF APLYSIA CALIFORNICA

Histamine (HA) is present in substantial quantities in all ganglia of Aplysia californica. Within the cerebral ganglia this amine is known to be concentrated in at least two identified neurons designated C-2 neurons. In this study a combination of chemical and enzymatic analyses was employed to provide evidence for the existence of a biochemical pathway for HA synthesis in ganglia and individual neurons of this marine mollusk. Examination of extracts of individual neurons dissected from ganglia organ-cultured in the presence of [³H]histidine showed that every neuron accumulated labelled histidine, but only the HA-containing C-2 neurons synthesized and stored labelled HA suggesting that the formation of HA in Aplysia could be catalyzed by the enzyme histidine decarboxylase (HDC). HDC activity was studied with a new microradiometric assay. Many of the properties of the molluscan HDC studied were found to correspond to the vertebrate enzyme. Enzyme activity was inhibited by α-hydrazino-histidine but unaffected by concentrations of α-methyldopa or by 5-(3,4-dihydroxycinnamoyl) salicylic acid which produced nearly complete inhibition of aromatic amino acid decarboxylase activity. HDC was measurable in nervous but not other Aplysia tissues assayed. All 5 major ganglia contained HDC activity which spanned a 15-fold range between the least and most active ganglia. Only 4 of the 13 nerve trunks assayed yielded measurable enzymic activity; these active nerves were associated with the cerebral ganglia which has the highest HDC activity of all measured ganglia. Of the numerous individual neurons assayed for HDC, only the C-2 cells showed measurable enzyme activity, about 25 pmol/cell/h or 70 μmol/g protein/h. Since the activity of HDC in the HA-containing neurons was at least three orders of magnitude larger than all other neurons assayed in the cerebral and other ganglia, these data appear to provide a direct metabolic basis for the selective presence of HA in these cells, and they indicate that the cellular presence of HDC provides a useful biochemical marker for the location of HA-rich neurons in Aplysia.     

Distribution of acid phosphatase and β-glucuronidase in the hypoplastic cerebellum of jaundiced Gunn rats

Summary Activities of acid phosphatase and -glucuronidase in the cerebella of young jaundiced (j/j) and non-jaundiced (j/+; control) Gunn rats were studied with the enzyme histochemical method. The cerebellum of j/+ rats showed high acid phosphatase activities in Purkinje cells and neurons in the cerebellar nuclei. In j/j rats, a number of neurons were lost and numerous microglialike cells with a high acid phosphatase activity appeared in the hypoplastic cerebellum. Although -glucuronidase activity was rarely detected in the control cerebellum, a high enzyme activity was observed associated with microglialike cells in j/j rats. The present results provide a cytological basis for the reported differential increase in the activities of these lysosomal enzymes in the j/j rat cerebellum.     

Synthesis of β‐Ketoamide Curcumin Analogs for Anti‐Diabetic and AGEs Inhibitory Activities

Two different series of novel β‐ketoamide curcumin analogs enriched in biological activities have been synthesized. The synthesized compounds were screened for their in vitro anti‐diabetic and AGEs inhibitory activities and exhibited potent to good anti‐diabetic and AGEs inhibitory activities. The molecular docking study was also performed with the α‐amylase enzyme.     

The rise and fall of pineal N-acetyltransferase in vitro: Neural regulation in the developing rat

In rats, the pineal gland has a rhythm in the activity of the enzyme, N-acetyltransferase (NAT), which is thought responsible for daily cycles of melatonin synthesis. Neonatal rat pineal glands, but not those of adult rats, have a single cycle that is observed in vitro during the first day of organ culture. The neural regulation of the cycle was investigated using neonatal rats with adult rats used for comparison. Prior treatment of rat pups with constant light did not abolish the cycle in vitro though it did abolish the in vivo rhythm. Removal of the superior cervical ganglia did not abolish the in vivo rhythm that was measured the first day after surgery, but ablation of the ganglia did abolish the rhythm if several days or more elapsed after surgery. Extirpation of the superior cervical ganglia abolished the in vitro NAT cycle in pup pineal glands as did the pharmacological equivalent, injection of 6-hydroxydopamine. Propranolol, a beta blocking agent, prevented the occurrence of the cycle in vitro.