Isolation and chemical characterization of Alzheimer's disease paired helical filament cytoskeletons: differentiation from amyloid plaque core protein.

The paired helical filaments (PHFs) of Alzheimer's disease were purified by a strategy in which the neurons and amyloid plaque cores of protein (APCP) were initially isolated. This was achieved by several steps of isocratic sucrose centrifugations of increasing molarity and a discontinuous isotonic Percoll density gradient. After collagenase elimination of contaminating blood vessels, lysis of neurons was produced by SDS treatment. The released PHF cytoskeletons were separated from contaminating APCP and lipofuscin by sucrose density gradient. A final step consisted in the chemical purification of highly enriched PHFs and APCP components via a formic acid to guanidine hydrochloride transition. PHFs and APCPs were fractionated by size exclusion HPLC and further characterized and quantitated by automatic amino acid analysis. We also present some of the morphological and immunochemical characteristics of PHF polypeptides and APCP. Our studies indicate that apart from differences in localization and morphology, PHF and APCP significantly differ in (a) chemical structure (peptide and amino acid composition); (b) epitope specificity (antiubiquitin, antitau, antineurofilament); (c) physicochemical properties (structural conformation in guanidine hydrochloride); and (d) thioflavine T fluorescence emission. These parameters strongly suggest important differences in the composition and, probably, in the etiopathology of PHF and APCP of Alzheimer's disease.

Inhibition of rat ovarian [3H]thymidine uptake by luteinizing hormone-releasing hormone agonists: a possible mechanism for preventing damage by cytotoxic agents.

To investigate the mechanism involved in luteinizing hormone-releasing hormone (LHRH) agonists' protective effects against chemotherapy-induced ovarian damage, female rats were either implanted with 1-mg pellets of LHRH agonist Zoladex (LHRHz) or sham operated. All rats were implanted with osmotic minipumps loaded with [3H]thymidine 48 h before sacrifice in diestrus. Ovaries were combusted in a biological material oxidizer. Tritiated water was recovered in a special cocktail, and ovarian tritiated thymidine uptake (3HTU) was calculated. In five experiments, LHRHz significantly reduced ovarian 3HTU. This was observed 5 days after implanting LHRHz pellets. Ovarian 3HTU correlated significantly with serum estradiol, LH, and ovarian and uterine weights. Autoradiography showed that almost all ovarian 3HTU is by granulosa cells. These data suggest that LHRHz suppresses ovarian mitotic activity. Since cytotoxic agents preferentially destroy rapidly dividing cells, our findings may represent a mechanism for ovarian protection.

Platelet-derived growth factor (PDGF)-signaling mediates radiation-induced apoptosis in human prostate cancer cells with loss of p53 function.

Platelet-derived growth factor (PDGF) signals a diversity of cellular responses in vitro, including cell proliferation, survival, transformation, and chemotaxis. PDGF functions as a "competence factor" to induce a set of early response genes expressed in G1 including p21WAF1/CIP1, a functional mediator of the tumor suppressor gene p53 in G1/S checkpoint. For PDGF-stimulated cells to progress beyond G1 and transit the cell cycle completely, progression factors in serum such as insulin and IGF-1 are required. We have recently shown a novel role of PDGF in inducing apoptosis in growth-arrested murine fibroblasts. The PDGF-induced apoptosis is rescued by insulin, suggesting that G1/S checkpoint is a critical determinant for PDGF-induced apoptosis. Because recent studies suggest that radiation-induced signal transduction pathways interact with growth factor-mediated signaling pathways, we have investigated whether activation of the PDGF-signaling facilitates the radiation-induced apoptosis in the absence of functional p53. For this study we have used the 125-IL cell line, a mutant p53-containing, highly metastatic, and hormone-unresponsive human prostate carcinoma cell line. PDGF signaling is constitutively activated by transfection with a p28v-sis expression vector, which was previously shown to activate PDGF alpha- and beta- receptors. Although the basal level of p21WAF1/CIP1 expression and radiation-induced apoptosis were not detectable in control 125-IL cells as would be predicted in mutant p53-containing cells, activation of PDGF-signaling induced expression of p21WAF1/CIP1 and radiation-induced apoptosis. Our study suggests that the level of "competence" growth factors including PDGF may be one of the critical determinants for radiation-induced apoptosis, especially in cells with loss of p53 function at the site of radiotherapy in vivo.

Thyroid injury, autoantigen availability, and the initiation of autoimmune thyroiditis.

Iodine depletion prevents disease and iodine repletion, which may cause thyroid cell injury by reactive oxygen intermediates, initiates disease in the Obese Strain chicken model of spontaneous autoimmune thyroiditis (AT). To examine the role of cell injury and autoantigen availability in AT induction we compared the immune responses that followed blunt trauma to the OS thyroid in the absence of iodine and the administration of normal dietary iodine in the absence of thyroid injury. Serum thyroglobulin concentrations were elevated following thyroid injury and the extensive thyroid infiltrates had high macrophage/CD4+, CD8+, B cell ratios consistent with an acute inflammatory response. The response was self limiting and undetectable in all animals 2 weeks later. Birds raised on a similar low iodine regimen were withdrawn from the regimen and given normal dietary iodine. Their thyroids showed no evidence of acute ultrastructural damage. The resulting early thyroid infiltrates had low macrophage/CD4+, CD8+, B cell ratios. Two weeks later these animals showed severe thyroid infiltration (43%) and after 4 weeks all animals had >90% infiltration. Thus, the presence or absence of thyroidal iodine, whether accompanied by injury or not, determined the nature and consequence of the immune response which argues against hypotheses that include obligatory injury at disease onset. Taken with previous work, this study suggests that iodination of an autoreactive thyroglobulin epitope is a requisite pathogenic action of iodine and supports the notion that in some organ-specific autoimmunity, a component of the dysregulation is associated with aberrant activity of the target tissue.

An animal model of pulmonary radiation fibrosis with biochemical, physiologic, immunologic, and morphologic observations.

An animal model of pulmonary radiation fibrosis was established, using male CBA/j mice. Both lungs of each mouse in one group (DL) were irradiated with two doses of 8.5 Gy each, separated by 30 days. A control group (CG) was sham-irradiated. There was a small but significant difference (P less than 0.03) in average breathing rate between DL and CG 27 weeks after the second irradiation which increased until the 34th week followed by a plateau. The accumulated hydroxyproline content of the irradiated mouse lung was 40% greater (P less than 0.02) than that of the sham-irradiated lung at 42 weeks and thereafter. Anticollagen antibodies assayed 52 weeks after irradiation by enzyme-linked immunosorbent assay were elevated by 49% in sera from the irradiated mice compared to sera from sham-irradiated mice. Mortality during the 52-week period following the second irradiation was low (13%) for both groups. Histological comparison of irradiated and control mouse lungs fixed under uniform inflation pressure indicated no significant differences. The model has unique features including an increase in collagen deposition, no acute changes attributable to radiation, a small but statistically significant abnormality in pulmonary function, an immunologic response to collagen, and low mortality.

Shigella dysenteriae 60R strain adheres to and invades tissue culture cells in the absence of virulence plasmid.

Evidence is presented that a high level Shiga toxin-producing strain Shigella dysenteriae 60R adheres to and invades the epithelial cell lines Hct8 and Henle 407. The invasive phenotype of S. dysenteriae 60R differs in four ways from the heretofore studied invasive Shigella phenotypes. First, S. dysenteriae 60R lacks the virulence plasmid characteristic of other invasive Shigella spp. and enteroinvasive Escherichia coli. Second, hybridization studies show that the known ipa genes are neither present in the chromosome nor in the small plasmid of 60R. Third, 60R adheres to and invades Hct8 and Henle 407 cells at 37 degrees C as well as at 30 degrees C. Fourth, the phenotype of adherence and invasion of 60R is remarkably stable, even during prolonged growth in laboratory media and storage.

In vivo and in vitro toxicity of phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces phospholipase C (PLC), a heat-labile hemolysin. Histopathological analysis of PLC-treated mice revealed that the primary target organs involved in PLC-induced toxicity were the liver and kidney. Mice treated i.v. with PLC demonstrated significant tubular epithelial necrosis of the kidney with hematuria, while when given i.p. they exhibited hepatonecrosis with cellular infiltration. Splenomegaly was also a consistent finding. Results from in vitro studies indicate that PLC is toxic for mouse peritoneal cells and human leukocytes.

New biochemical insights to unravel the pathogenesis of Alzheimer's lesions.

Purification of amyloid plaque core proteins (APCP) from Alzheimer's disease brains to complete homogeneity and in high yield permitted its chemical fractionation and characterization of its components. APCP is mainly made of beta-amyloid (beta A) and an assortment of glycoproteins (accounting for 20%) rich in carbohydrates compatible with N- and O-linked saccharides. When added to tissue culture of sympathetic and sensory neurons APCP and beta A inhibited neuritic sprouting, a reversible phenomenon at low doses. Higher concentrations of both substances kill the neurons in culture. APCP is significantly more toxic than beta A, suggesting the minor components may play an important role in increasing the toxicity of beta A. If the observed toxic effects of APCP in situ are occurring in vivo during the course of AD, then the accumulation of these extracellular proteins could be largely responsible for some of the neuronal death observed in this neuropathology.

Clara cell adenomas of the mouse lung. Interaction with alveolar type 2 cells.

Multiple pulmonary adenomas were induced in the offspring of pregnant Swiss-Webster mice by transplacental exposure to ethylnitrosourea (ENU) on the 15th day of gestation. Development and growth of tumors were followed for up to a year after birth. Morphologic assessment indicated that the majority of adenomas were of Clara-cell origin and were relatively normal on the basis of structural features. Histochemical studies, utilizing nitroblue tetrazolium reductase activity as a marker for normal Clara cells demonstrated that the Clara-cell-derived tumors possessed nearly normal enzyme activity. Microscopic studies of the tumors and adjacent parenchyma revealed a unique Type 2 cell response to the presence of Clara-cell adenomas occurring in the alveoli beyond the margins of the tumor. Otherwise normal-appearing Type 2 cells, in a narrow zone around the Clara-cell tumors, accumulated large amounts of surfactantlike osmiophilic lamellar material within cytoplasmic vacuoles as early as 30 days after birth. These changes were clearly a Clara-cell-tumor-related response, and not seen in association with other non-Clara-cell adenomas of the same lung. Furthermore, the alterations occurred exclusively in Type 2 cells. The extent of Type 2 cell change was correlated with tumor size and age. Autoradiographic studies with tritiated choline showed marked incorporation of the labeled precursor by the altered Type 2 cells. By electron microscopy, these inclusions were membrane-limited and contained osmiophilic lamellar structures similar to lamellar bodies in normal Type 2 cells. Because these Clara cell adenomas may act as a concentrated focus of normal Clara cells, the alterations seen in Type 2 cells may reflect an amplification of a normal interaction between bronchiolar Clara cells and alveolar Type 2 cells in the centriacinar and juxtabronchiolar alveoli.

Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers.

Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.

Beta-adrenergic regulation of secretion from Clara cell adenomas of the mouse lung.

Ethylnitrosourea-induced pulmonary adenomas of the mouse have been reported as being predominantly Clara cell in origin. The response of these tumor cells in vivo to the secretory agonist, isoproterenol (10 mg/kg) and the antagonist, propranolol (2.0 mg/kg) 1 hour after intraperitoneal injection into 120-day-old tumor-bearing mice was examined. Ultrastructural morphometry was used to quantitate the secretory response of tumor cells by measuring the volume density of the secretory granules. In the intact animal, isoproterenol stimulated secretion in the Clara cell adenomas (40% decrease in volume density with no change in surface to volume ratio of granules), while propranolol prevented this effect. In addition, beta-adrenergic receptors on isolated tumor cells were demonstrated by radioligand-binding assay by using [125I]iodocyanopindolol (ICYP). Scatchard analysis of data derived from whole cells indicates a maximum receptor-binding capacity of 27 fmoles/mg of protein and a KD of 0.029 nM. Isoproterenol displacement of ICYP binding yields an IC50 of 8 X 10(-7) M and a calculated KD of 3.36 X 10(-7) M. The beta 2 identity of these receptors was determined by utilizing the relatively specific beta 1 and beta 2 antagonists practolol and ICI-118,551, respectively. Practolol failed to displace more than 30% of ICYP binding even at 100 microM, while ICI-118,551 displacement of ICYP yielded a linear Hofstee plot (r = 0.93) and a KD of 5.04 X 10(-9) M. These findings suggest that the secretory activity of Clara cell-like pulmonary adenomas is under beta-adrenergic control similar to that of normal bronchiolar Clara cells.

Cadmium-induced acute lung injury: compromised repair response following thyroidectomy.

The role of thyroid hormone in the pulmonary repair process following chloride-induced acute injury, was assessed in the present study. Thyroidectomized (Thyx), male Sprague-Dawley rats were exposed by inhalation to cadmium chloride aerosol (CdCl2, 10 mg/m3). Rats were sacrificed 1 hr after [3H]thymidine (3H-T) injection at intervals up to 10 days after exposure. Thyroidectomy, followed by CdCl2, produced earlier and more severe acute injury in the form of alveolar hemorrhage edema and hyaline membrane formation, than CdCl2 alone. However, Type 2 cell hyperplasia was markedly reduced in this group of rats compared with CdCl2 controls. Uptake of 3H-T by Thyx-CdCl2 lung tissue was only 40% of that measured in CdCl2 controls. Autoradiographic studies indicated that Type 2 cell labeling was less than 66% of controls up to 3 days after exposure. Cells obtained by lung lavage of Thyx-CdCl2 rats were reduced in number up to 60% with respect to controls, during the first week after exposure. Additionally, the activities of lung antioxidant enzymes (glucose-6-phosphate dehydrogenase, superoxide dismutase, and glutathione peroxidase were significantly inhibited (45-55%) throughout the experiment in Thyx-CdCl2 animals compared with normal rats. In summary, thyroidectomy impairs the repair response in CdCl2 lung damage by enhancing Type 2 cell damage, reducing Type 2 cell proliferation, altering alveolar macrophage populations, and depressing antioxidant defense systems.

Acute lung injury induced by cadmium aerosol. I. Evolution of alveolar cell damage.

Following exposure to an aerosol of 0.1% (.005 M) cadmium chloride, rat lungs were examined at 6 hours and 1, 2, 3, 4, 7, and 10 days. By light microscopy, damage was multifocal and centered about respiratory bronchioles. Ultrastructurally, there was Type 1 cell edema with frequent loss of surface plasma membranes during the first 24 hours. After 2 days, the number of Type 2 cells had markedly increased, and by 3 days the damaged alveoli were lined by plump cuboidal cells closely resembling Type 2 cells. By 4 days, these cells were flatter, the change being more marked by 7 days; by 10 days, the cells had regained the appearance of Type 1 cells through loss of osmiophilic bodies and superficial microvilli. We conclude that CdCl2 damages Type 1 cells, which are then replaced by proliferation of Type 2 cells. These cells lose their osmiophilic bodies and flatten out to replace the lost Type 1 cells, the process being almost complete by 10 days after the injury. This pattern resembles the injury caused by NO2, O3, and O2.

Cellular proliferation induced in the lung by cadmium aerosol.

Rats were exposed to a polydispersed aerosol of 0.1 per cent cadmium chloride in physiologic saline for a single period of 2 hours, and the evolution of damage was followed for a 10-day period. Control animals were unexposed rats and rats exposed to an aerosol of physiologic saline. Wet lung weight doubled by the fourth day in animals exposed to cadmium chloride, but not in the control groups. Apart from a transient increase in fluid content at 1 day, there was no evidence that the lung weight gain was due to inflammatory edema. Uptake of [3H]thymidine markedly increased during the first four days after exposure to cadmium chloride. Light autoradiography showed that [3H]thymidine labeling occurred almost exclusively in Type II alveolar cells at 1 day: later, the label appeared in interstitial cells and cells lying free within alveoli. These changes correlated well (r=0.70) with the increase in deoxyribonucleic acid on the fourth day after exposure. Uptake of [3H]thymidine and deoxyribonucleic acid content in rats exposed to cadmium chloride showed a significant difference from the control groups (P less than 0.001). These experiments demonstrate that exposure to cadmium chloride aerosol evokes a wave of cellular proliferation in rat lung. This finding is of interest, because heavy industrial exposure to cadmium in humans is known to be associated with severe physiologic impairment and anatomic damage.

The evolution of biochemical damage in the rat lung after acute cadmium exposure.

Rats were exposed once to a polydisperse aerosol of 0.005 M cadmium chloride for 2 hours. Controls were saline-exposed rats and unexposed rats. Total extractable lipid, malate, lactate, isocitrate, and glucose-6-phosphate dehydrogenases were measured on the organelle-free cytosol from homogenized lungs at periods up to 10 days after exposure. The wet weight, dry weight, ribonucleic acid, and deoxyribonucleic acid content of the lungs were also determined. Ultrastructural appearances were studied at the same time intervals in a separate experiment. Total lipid content, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase activities showed approximate doubling by the fourth day after exposure, this change coinciding with doubling of wet lung weight and total lung deoxyribonucleic acid content. Malate dehydrogenase activity showed a high peak 1 hour after exposure before decreasing to follow the pattern of lactate and glucose-6-phosphate dehydrogenase. The changes found may be a nonspecific reaction to lung injury, comparable with oxygen, ozone, and nitrogen dioxide. These results in Type II cell proliferation, which would enhance glucose-6-phosphate dehydrogenase content and resistance to peroxidation. Lipid accumulation could be a similar response. However, the initial elevation in malate dehydrogenase activity is more in keeping with a specific mitochondrial injury (with some ultrastructural support), perhaps with leakage of mitochondrial enzymes. This suggests that there may be more than one mechanism at work in the injury. It is significant that despite the marked structural and functional changes, none of the rats died after the exposure, which makes the injury a model worthy of further investigation.

Vitamin D3 receptor expression in N-ethylnitrosourea-induced mouse pulmonary adenomas.

An expanded role for vitamin D (1 alpha,25-(OH)2D3) in mammalian systems has been suggested by recent evidence that its receptor (vitamin D receptor [VDR]) is present not only in classical target organs, but in a variety of normal tissues and organs, tumor tissues, and cancer cell lines. Vitamin D is involved not only in the regulation of calcium homeostasis and bone metabolism, but in the regulation of cell proliferation, differentiation, and immune responses. The role vitamin D may play in normal lung growth, development, and maintenance is unknown. Likewise, its part in lung tumorigenesis is unclear. The present study examined VDR binding activity and VDR expression in normal mouse lung and ethylnitrosourea-induced lung adenomas. Binding of 1 alpha,25-(OH)2D3 was specific and saturable over the concentration range of 0.01 to 0.50 nM, with an affinity (Kd) of 0.93 x 10(-10) M and a total binding capacity (Bmax) of 22 fmol/mg of protein. Scatchard analysis yielded a convex curve, which suggests positive receptor cooperativity. The calculated Hill coefficient equals 1.69, at a receptor concentration of 0.4 nM, consistent with dimerization of the receptor. Western blot analysis showed the presence of 60 kD VDR protein in tumor homogenates, while Northern blot analysis detected the 4.4 kb VDR mRNA in tumor tissue preparations. Immunohistochemistry and in situ hybridization revealed that both adenomatous Clara cells and normal bronchiolar epithelial Clara cells expressed VDR, with the receptor protein present in their nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor-induced apoptosis in A431 cells can be reversed by reducing the tyrosine kinase activity.

A431 cells overexpress epidermal growth factor receptors (EGF-Rs) and are inhibited by EGF. We show that treatment of A431 cells with 10 nM EGF induced a 15-fold increase in EGF-R autophosphorylation, leading to inhibition of cell proliferation and morphological features of apoptosis. However, at a lower concentration of EGF (0.01 nM), there is a 2-fold increase in EGF-R autophosphorylation and increased cell proliferation when compared to untreated cells. EGF treatment is associated with increased expression of c-myc and decreased expression of mutant p53 and p21/WAF protein. When A431 cells were simultaneously treated with 10 nM EGF and EGF-R antibody, there was a significant reduction in EGF-R autophosphorylation that was associated with increased cell proliferation. Based on these results, we postulate that overexpression of EGF-R could allow for selective growth advantage for tumor cells in the presence of normal or decreased ligand availability. However, excessive ligand binding would result in deregulated growth signaling, leading to growth inhibition and programmed cell death.

Identification of tissue-specific DNase I hypersensitive sites in the rabbit flavin-containing monooxygenase form 2 gene.

Previous studies demonstrated that the flavin-containing monooxygenases (FMO) are expressed in a tissue-specific manner. To begin an elucidation of mechanisms regulating this expression pattern, genomic clones for rabbit FMO2 were isolated and characterized. Two clones were isolated from a lambda EMBL3 genomic library and shown to span approximately 30 kilobase pairs but to contain only about 800 base pairs of coding information. Primer extension analysis was used to map the transcription start site, extending the previously published cDNA sequence by 17 base pairs. The FMO2 promoter does not utilize a classical TATA box, nor are HTF islands present (DNA domains rich in cleavable sites for 5-methyl-cytidine/guanosine-sensitive restriction enzymes). Rather, homology with promoters controlled by initiator elements is observed. Previous studies demonstrated FMO2 expression in rabbit pulmonary Clara and type II cells [Overby, Nishio, Lawton, Plopper, and Philpot: Exp. Lung Res. 18, 131, (1992)]. In the present study, a highly enriched Clara/type II cell population was prepared, and the FMO2 gene was analyzed for DNase I-hypersensitive and methylated regions. These data were then contrasted with those obtained from a similar analysis of the nonexpressed, hepatocyte FMO2 gene. No difference in the methylation status was observed. However, Clara/type II cell-specific DNase I-hypersensitive sites are located within the promoter region of the FMO2 gene. Thus, tissue-specific transcription factors likely are more prominent than methylation in regulating FMO2 expression. Consistent with this observation, both polyomavirus enhancer activator 3 (E26 transformation specific) and thyroid transcription factor 1 consensus sequences are present within the tissue-specific DNase I-hypersensitive domain.

Adenosine-induced apoptosis in chick embryonic sympathetic neurons: a new physiological role for adenosine.

1. A newly found action of adenosine in neurons, which may have an important physiological function in the growth and development of the sympathetic nervous system, is described. Adenosine (1-100 microM) inhibited neurite outgrowth within the first 24 h and killed about 80% of sympathetic neurons supported by nerve growth factor over the next 2 days in culture. Neurons supported by excess KCl, forskolin or phorbol 12,13-dibutyrate were equally susceptible to the toxic actions of adenosine. Inosine, guanosine or hypoxanthine (all 100-300 microM) were without effect on neuronal growth and survival. 2. Specific agonists of adenosine A1 and A2 receptors were not neurotoxic, and toxic effects of adenosine were not antagonized by aminophylline. These results rule out involvement of adenosine receptors and the adenylyl cyclase-cAMP signalling system in neurotoxic actions of adenosine. 3. Adenosine toxicity was prevented by inhibitors of the adenosine membrane transporter, suggesting an intracellular site of action of adenosine. 4. Inhibitors of adenosine deaminase dramatically facilitated the toxic action so that physiologically relevant concentrations of adenosine were neurotoxic. 5. Adenosine kinase activity of sympathetic neurons was dose-dependently inhibited by 5'-iodotubercidin (3-100 nM). 5'-Iodotubercidin (100 nM) completely protected neurons against toxicity of adenosine plus adenosine deaminase inhibitors. These results provide convincing evidence that phosphorylation of the nucleoside is an essential requirement for initiation of adenosine toxicity. 6. Sympathetic neurons were successfully rescued from the lethal effects of adenosine deaminase inhibitor plus adenosine by uridine or 2-deoxycytidine, but not by nicotinamide or 2-deoxyguanosine, suggesting that depletion of pyrimidine nucleotides by phosphorylated adenosine compounds and consequent inhibition of DNA synthesis produces neuronal death. 7. DNA fragmentation, assessed by the fluorescent dye bisbenzimide and by the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labelling) method, indicated that neuronal death induced by adenosine was apoptotic. 8. We conclude that adenosine deaminase and adenosine kinase play an important role in the metabolism of intracellular concentrations of adenosine and thereby regulate the growth and development of sympathetic neurons. Our study highlights, for the first time, the importance of adenosine as a mediator of programmed cell death of neurons supported by nerve growth factor.