Differential effects of various cAMP derivatives on the morphological and electrical maturation of neuroblastoma X glioma hybrid cells

The influence of adenosine 3′: 5′-cyclic monophosphate (cAMP) and some of its derivatives on the morphological differentiation and on the expression of electrical activity was investigated in neuroblastoma X glioma hybrid cells. This permanent cell line constitutes a well established culture system for studying neuronal properties in vitro. cAMP (1 mM) caused cell death. With 8-bromo-cAMP (0.1–1 mM) present giant multinuclear cells appeared, which were more obvious at 0.1 than at 1 mM 8-bromo-cAMP. 8-p-chlorophenylthio-cAMP (0.1–1 mM) induced an extension of neurites. These cellular processes were comparable to those elicited in the presence of db-cAMP (1 mM). However, only the cells treated with db-cAMP, but not those exposed to 8-p-chlorophenylthio-cAMP, were found to generate action potentials upon electrical stimulation. Neither dexamethasone nor carboxymethylcellulose, nor 8-bromo-cAMP could elicit the formation of processes in hybrid cells.     

Relationship between cyclic AMP, microtubule organization, and mammalian cell shape : Studies on Chinese hamster ovary cells and their variants

Treatment of Chinese hamster ovary cells with N⁶,2′-O-dibutyryl adenosine cyclic 3′,5′-monophosphate (db-cAMP) and hormones converts their shape from a knobbed, epithelial-like morphology to a smooth fibroblast-like form. Ultrastructural studies demonstrate an increased number of microtubules and their arrangement in parallel array along the long axis of the cell after treatment with these agents. Although an epithelial-like variant, when treated with db-cAMP, shows an increase in the number of microtubules; these microtubules remain in a disorganized nonparallel array. The numerous long microtubules which are already present in a fibroblast-like variant become further elongated when the cells are treated with db-cAMP. These experiments establish the relationship between cAMP level, assembly and organization of microtubules, and cell shape in cultured Chinese hamster cells.     

Reversible inhibition of cAMP-induced axon formation in neuroblastoma cells by concanavalin a and vinblastine : Relationship to microtubules and cytotoxicity

The formation of axons induced by dibutyryl-adenosine 3′,5′-cyclic monophosphate (db-cAMP) in neuroblastoma cells was inhibited by concanavalin A (ConA) and vinblastine. These compounds also caused the retraction of existing axons. After removal of ConA or vinblastine, addition of db-cAMP again resulted in axon formation. The cytotoxicity of ConA and vinblastine for neuroblastoma cells was reduced when cell multiplication was inhibited by db-cAMP. Linearly growing normal fibroblasts were also more sensitive to the cytotoxic effect of ConA than confluent non-multiplying fibroblasts. The effects of ConA and vinblastine were additive both in their effects on axon formation and cytotoxicity. Wheat germ agglutinin (WGA) and lumicolchicine did not affect axon formation or reduce cell viability. It is suggested that ConA bound to the cell surface can interfere with the assembly of cytoplasmic microtubules involved in axon formation and cell division.     

The chronic effects of db-cAMP on cell cycle parameters and cell size in asynchronous culture of Chinese hamster ovary cells

N⁶,O^2′-dibutyryl adenosine 3′,5′-cyclic-phosphate (db-cAMP) has been shown to convert Chinese hamster cells of ovarian origin (CHO-K₁) from compact, randomly oriented cells growing in multilayers to elongated fibroblast-like cells which grow in monolayers. This compound also has been reported to have a variety of effects on the cell cycle. Most such studies have employed synchronized cells to determine cell cycle effects, and consequently have been limited to the short-term effects of the compound. We have looked for chronic effects on the cell cycle in cultures exposed continuously to db-cAMP from the initiation of the cultures until they had reached or approached the plateau phase. This was done by combined autoradiography and Feulgen microspectrophotometry plus measurements of the protein content of mitotic cells to detect any influence on cell size. The overall results were that continuous exposure to db-cAMP had at most only minor effects on the cell cycle and cell size when the culture medium was renewed daily. Somewhat greater effects were found on plateau-phase cells in cultures in which the medium was not renewed. In this case fewer cells appeared to remain in the cell cycle in the cultures with db-cAMP. Comparison with our earlier results with Chinese hamster V79 cells led to the conclusions that cell cycle parameters and cell size at mitosis were less altered during culture growth in CHO cells, but that CHO cells seemed to be less able to maintain cells in the cell cycle in crowded cultures.     

A correlation between membrane glycopeptide composition and losses in concanavalin a agglutinability induced by db-cAMP in Chinese hamster ovary cells

A double-label method, employing [¹⁴C] - and [³H] -fucose, has been used to compare the carbohydrate components of surface glycoproteins from four different sub-clones of Chinese hamster ovary cells grown in the presence or absence of either 3′: 5′-dibutyryl cyclic AMP (db-cAMP), 3′: 5′-cyclic AMP (cAMP) or a phosphodiesterase inhibitor SQ 20009. Following growth in one or more of these drugs, a number of these sub-clones showed a fairly small, but consistent reduction in the amount of the more rapidly eluting fucopeptides that could be isolated from the plasma membrane and a corresponding increase in lower molecular weight components as determined by Sephadex G-50 chromatography. This apparent decrease in the size of surface fucopeptides was related to a reduced sialic acid content of a class of surface glycopeptides isolated from the treated cells. This surface change was always correlated with a loss of concanavalin A (ConA)-mediated agglutinability. However, this surface change was not invariably associated with the drug-induced morphological transition towards a more fibroblast-like form. More-over, the sialic acid-rich glycopeptides bound only poorly to ConA affinity columns and were probably not therefore the lectin receptors. Double-label experiments have shown that upon addition of db-cAMP to the cells, existing glycopeptides are apparently unmodified but rather new components reaching the cell surface have a reduced amount of sialic acid associated with them. We propose that the loss in lectin-induced agglutinability and the reduction in glycopeptide size are related phenomena resulting from a primary change in cell surface chemistry.     

The ultrastructure of process formation following treatment with db-cAMP of a Chinese hamster ovary X Chinese hamster brain cell hybrid

Scanning (SEM) and transmission (TEM) electron microscopy studies were performed on a hybrid, resulting from the Sendai-virus fusion of a Chinese hamster ovary (CHO) Glycine A (Gly⁻A) auxotrophic mutant cell [1,2] with a freshly-biopsied suspension of Chinese hamster cerebral cortex cells. In normal growth medium the hybrid differs from the CHO parental cell in displaying a squamous, polygonal, epithelioid appearance with sparse microvilli and ribosome-filled knobs (blebs). Slender filopodia, which sometimes reach a length of 25 μm, extend from interphase cells. Bundles of microfilaments (6 nm diameter) are observed closely associated with the cell membrane and the perinuclear region, arranged more or less in parallel to the glass substrate. The untreated hybrid has a relatively unpatterned arrangement of microtubules and reveals desmosomes at points of cell contact. When treated with N⁶,O²-dibutyryl adenosine 3′:5′-cyclic monophosphoric acid (db-cAMP) plus the Synergist testololactone, the response of the hybrid differs markedly from the fibroblastic habitus assumed by CHO [3, 4, 5]. The hybrid cells become stellate, forming processes or cytoplasmic extensions which radiate from a central, microvillus-covered, rounded, cell body. The arborizing processes number 2–8 per cell and form a contiguous network between cells of a colony. Desmosomes are seen at these points of process-to-cell body junctions. Parallel microtubules, 10 nm filaments, and 6 nm microfilaments, as well as organelles of the unstimulated cytoplasm such as free ribosomes, lipid granules, mitochondria, rough ER, and myelin figures are present in the nerve-like extensions. Knobs disappear completely following cAMP treatment. On removal of the db-cAMP, disappearance of the processes is apparent in 1–2 h so that the cell returns to its original morphology. This reversal is accompanied by ruffling activity at the cell borders. The central, rounded portion of the cell returns to the former flattened state somewhat more slowly. These studies demonstrating a cAMP-induced change in morphology and microtubule arrangement, produced in CHO X brain cell hybrids support the previous proposals that: (1) cAMP action is necessary for organization of cellular microtubules to form a pattern; (2) this pattern is a function of the cellular differentiation state and is determined by genetic or epigenetic factors.     

Guanosine 5′-triphosphate inhibits growth and stimulates differentiated functions in B16 melanoma cells

Exogenous guanosine 5′-triphosphate (GTP) at a concentration of 0.5 mM causes in vitro growth inhibition and induces morphological and biochemical differentiation of B16 melanoma cells. After two days in the presence of GTP, cell proliferation is markedly reduced. Cessation of cell proliferation is followed by the extension of numerous dendrite-like processes and marked increase in melanin production. Other nucleotides such as guanosine 5′-diphosphate (GDP), guanosine 5′-monophosphate (GMP), guanosine 3′:5′-cyclic-monophosphoric acid (cGMP) or adenosine 5′-triphosphate (ATP) have little or no effect on cell morphology or melanin production in B16 melanoma cells, although these compounds retard cell proliferation similar to GTP. These findings are discussed in light of a possible relationship between cell proliferation and differentiation.     

Effect of PCBs on androgen production by suspension of adult rat Leydig cells in vitro

The effects of PCBs (mixture of 2, 3, 4, 5-tetra; 2, 2′, 4, 5, 5′-penta; 2, 2′, 3, 3′, 6, 6′-hexa and 2, 2′, 3, 3′, 4, 4′, 5, 5′-octa congeners) on androgen production were investigated by suspension of Leydig cells from adult rat testis. hCG-stimulated androgen production was significantly inhibited by PCBs while progesterone level was not affected. Progesterone supported testosterone production was also decreased by PCBs, while conversion of androstenedione to testosterone was unchanged. These results suggest that the activity of microsomal enzyme C21 side-chain cleveage P450 was decreased by PCB treatment of Leydig cells in vitro.     

Regulation of DNA synthesis by guanosine-5′-diphosphate, cyclic guanosine-3′,5′-monophosphate, and cyclic adenosine-3′,5′-monophosphate in mouse lymphoid cells

The effect of various adenine and guanine nucleotides and nucleosides on DNA synthesis was studied in various types of mouse lymphoid cells. Two out of the ten compounds tested, namely guanosine-5′-diphosphate (GDP) and cyclic guanosine-3′,5′-monophosphate (cGMP) increased the thymidine incorporation into the DNA of the spleen cells and counteracted completely or partially the inhibitory action of cyclic adenosine-3′,5′-monophosphate (cAMP) on spleen cells stimulated by various B or T cell mitogens. GDP seems to act preferentially on thymus cells while cGMP acts better on bone marrow cells. The possible significance of the results for the mechanism of the mitogenic signal is discussed.     

The effect of serum on lipid synthesis and on the expression of oligodendrocyte marker-enzymes in oligodendrocyte-enriched glial cells in culture

Glial cells were isolated from the cerebra of 7-day old rats and the effect of serum on the development of these cells in culture was studied. The activities of the oligodendrocyte marker-enzymes, 2′3′-cyclic nucleotide 3′-phosphodiesterase and glycerol 3-phosphate dehydrogenase and the synthesis of the myelin-associated sulpholipid, sulphatide, were used to monitor the differentiation of these cells in vitro. The results indicate that serum: (i) represses lipogenesis, cholesterogenesis and sulphatide synthesis, (ii) lowers the expression of 2′3′-cyclic nucleotide 3′ phosphodiesterase and glycerol 3-phosphate dehydrogenase but not of lactate dehydrogenase and (iii) thus impairs the differentiation of oligodendrocytes.     

On the discovery of interferon-inducible, double-stranded RNA activated enzymes: the 2'-5'oligoadenylate synthetases and the protein kinase PKR

The demonstration that double-stranded (ds) RNA inhibits protein synthesis in cell-free systems prepared from interferon-treated cells, lead to the discovery of the two interferon-induced, dsRNA-dependent enzymes: the serine/threonine protein kinase that is referred to as PKR and the 2′,5′-oligoadenylate synthetase (2′,5′-OAS), which converts ATP to 2′,5′-linked oligoadenylates with the unusual 2′-5′ instead of 3′-5′ phosphodiesterase bond. We raised monoclonal and polyclonal antibodies against human PKR and the two larger forms of the 2′,5′-OAS. Such specific antibodies proved to be indispensable for the detailed characterization of these enzyme and the cloning of cDNAs corresponding to the human PKR and the 69–71 and 100 kDa forms of the 2′,5′-OAS. When activated by dsRNA, PKR becomes autophosphorylated and catalyzes phosphorylation of the protein synthesis initiation factor eIF2, whereas the 2′-5′OAS forms 2′,5′-oligoadenylates that activate the latent endoribonuclease, the RNAse L. By inhibiting initiation of protein synthesis or by degrading RNA, these enzymes play key roles in two independent pathways that regulate overall protein synthesis and the mechanism of the antiviral action of interferon. In addition, these enzymes are now shown to regulate other cellular events, such as gene induction, normal control of cell growth, differentiation and apoptosis.     

Effects of dibutyryl cyclic AMP and prostaglandin E1 on cultured human glioma cells

Dibutyryl cyclic AMP (db-cAMP) and prostaglandin E₁ (PGE₁) induced morphological alterations in cultured human glioma cells (138 MG). Cells in serum-free medium, treated with db-cAMP (1 mM) or PGE₁ (10μg/ml), within 1–3 h showed multiple thin processes resembling those of normal glial cells. These processes increased in size during a 24 h incubation. In serum-containing medium the appearance of cells with multiple processes was delayed. The induced morphological alterations were reversible upon exchange with fresh serum-containing but not with serum-deprived medium. Actinomycin D (5 μg/ml) did not prevent the changes induced by PGE₁ or db-cAMP. Inhibition of protein synthesis with cycloheximide (10 μg/ml) did not arrest the initial (1–3 h) changes in morphology but blocked further growth of the processes on prolonged incubation. Vinblastine sulphate (0.1 μg/ml) completely inhibited the alterations induced by PGE₁ or db-cAMP.     

Effect of Naturally Occurring Flavonoids on Lipid Peroxidation and Membrane Permeability Transition in Mitochondria

The ability of eight structurally related naturally occurring flavonoids in inhibiting lipid peroxidation and mitochondrial membrane permeability transition (MMPT), as well as respiration and protein sulfhydryl oxidation in rat liver mitochondria, was evaluated. The flavonoids tested exhibited the following order of potency to inhibit ADP/Fe(II)-induced lipid peroxidation, estimated with the thiobarbituric acid assay: 3′-O-methyl-quercetin > quercetin > 3,5,7,3′,4′-penta-O-methyl-quercetin > 3,7,3′,4′-tetra-O-methyl-quercetin > pinobanksin > 7-O-methyl-pinocembrin > pinocembrin > 3-O-acyl-pinobanksin. MMPT was estimated by the extent of mitochondrial swelling induced by 10 μM CaCl₂ plus 1.5 mM inorganic phosphate or 30 μM mefenamic acid. The most potent inhibitors of MMPT were quercetin, 7-O-methyl-pinocembrin, pinocembrin, and 3,5,7,3′,4′-penta-O-methyl-quercetin. The first two inhibited in parallel the oxidation of mitochondrial protein sulfhydryl involved in the MMPT mechanism. The most potent inhibitors of mitochondrial respiration were 7-O-methyl-pinocembrin, quercetin, and 3′-O-methyl-quercetin while the most potent uncouplers were pinocembrin and 3-O-acyl-pinobanksin. In contrast 3,7,3′,4′-tetra-O-methyl-quercetin and 3,5,7,3′,4′-penta-O-methyl-quercetin showed the lowest ability to affect mitochondrial respiration. We conclude that, in general, the flavonoids tested are able to inhibit lipid peroxidation on the mitochondrial membrane and/or MMPT. Multiple methylation of the hydroxyl substitutions, in addition to sustaining good anti-lipoperoxidant activity, reduces the effect of flavonoids on mitochondrial respiration, and therefore, increases the pharmacological potential of these compounds against pathological processes related to oxidative stress.     

Microbial synthesis of 2,6-diaminopurine nucleosides

2,6-Diaminopurine nucleosides are used as pharmaceutical drugs or prodrugs against cancer and viral diseases.

The synthesis of 2,6-diaminopurine riboside, -2′-deoxyriboside, -2′,3′-dideoxyriboside and -arabinofuranoside was efficiently carried out by transglycosylation using bacterial whole cells as biocatalysts. The preparation of 2,6-diaminopurine-2′,3′-dideoxyriboside catalysed by whole cells is here reported for the first time.     

Pyridoxal 5′-phosphate related changes in retention of 1,25-dihydroxy vitamin D-receptor ligands in rat intestinal mucosa cell nuclei

After feeding rats a vitamin B-6-deficient diet, we observed a decrease in pyridoxal 5′-phosphate concentrations in intestinal mucosa cells to 32 and 48% of control in cytoplasm and cell nuclei, respectively. Correlation analysis suggested that there were two pyridoxal 5′-phosphate pools in the nuclei: a “mobile” pool (equivalent to about 5% the concentration of the cytoplasmic pyridoxal 5′-phosphate), and a “stable” pool, which was independent of cytoplasmic fluctuations of pyridoxal 5′-phosphate (about 9 pmol pyridoxal 5′-phosphate/mg DNA). Reduction in pyridoxal 5′-phosphate content in the cells of vitamin B-6-deficient animals was accompanied by a substantial increase in 1,25-dihydroxyvitamin D-receptor ligand concentration in the cell nuclei (76.6 ± 19.7 vs 762 ± 291 fmol/mg DNA, mean ± SEM). The degree of 1,25-dihydrovitamin D accumulation in the nuclei appeared to be an exponential function of the “mobile” nuclear pyridoxal 5′-phosphate concentration. Semilogarithmic transformation of the data yielded a straight line, representing an inverse correlation between the cytoplasm-related nuclear pool of pyridoxal 5′-phosphate and the logarithm of the 1,25-dihydroxyvitamin D concentration in the nuclei (r=−0.95). These data suggest that pyridoxal 5′-phosphate may be related to 1,25-dihydroxyvitamin D retention in the nuclei, possibly through interaction of the pyridoxal 5′-phosphate with the vitamin D receptor protein in the nuclei.     

Further farnesyl-homogentisic acid derivatives from Otoba parvifolia

The seeds of Otoba parvifolia contain three novel compounds apparently derived from homogentisic acid, rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid and its acetate as well as rel-(1′R,4′S,5′R)-2-(1′-farnesyl-4′,5′-dihydroxy-2′-oxocyclohexan-1′-yl)-acetic acid δ-lactone. The structure of an additional isolate, previously described as 2-(1′-farnesyl-2′-hydroxy-5′-oxocyclohex-3′-en-1′-yl)-acetic acid γ-lactone was revised to rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid δ-lactone.     

Synthesis of (+/−)–1′-Aza-carbocyclic-pyrimidine-2′,3′-dideoxynucleoside analogues as potential anti-HIV agents

1′-Aza-carbocyclic-2′, 3′-dideoxyuridine, 3′-deoxythymidine and 2′, 3′-dideoxycytidine were synthesized from 1-aminopyrrolidine intermediate 13 and evaluated as anti-HIV agents in MT-4 cells.     

Isolation, structural elucidation, and partial synthesis of lutein dehydration products in extracts from human plasma

All-E-(3R,6′R)-3-hydroxy-3′,4′-didehydro-β,γ-carotene (anhydrolutein I) and all-E-(3R,6′R)-3-hydroxy-2′,3′-didehydro-β,ε-carotene (2′,3′-anhydrolutein II) have been isolated and characterized from extracts of human plasma using semipreparative high-performance liquid chromatography (HPLC) on a C₁₈ reversed-phase column. The identification of anhydroluteins was accomplished by comparison of the UV-Vis absorption and mass spectral data as well as HPLC-UV-Vis-mass spectrometry (MS) spiking experiments using fully characterized synthetic compounds. Partial synthesis of anhydroluteins from the reaction of lutein with 2% H₂SO₄ in acetone, in addition to anhydrolutein I (54%) and 2′,3′-anhydrolutein II (19%), also gave (3′R)-3′-hydroxy-3,4-dehydro-β-carotene (3′,4′-anhydrolutein III, 19%). While anhydrolutein I has been shown to be usually accompanied by minute quantities of 2′,3′-anhydrolutein II (ca. 7–10%) in human plasma, 3′,4′-anhydrolutein III has not been detected. The presence of anhydrolutein I and II in human plasma is postulated to be due to acid catalyzed dehydration of the dietary lutein as it passes through the stomach. These anhydroluteins have also been prepared by conversion of lutein diacetate to the corresponding anhydrolutein acetates followed by alkaline hydrolysis. However, under identical acidic conditions, loss of acetic acid from lutein diacetate proceeded at a much slower rate than dehydration of lutein. The structures of the synthetic anhydroluteins, including their absolute configuration at C(3) and C(6′) have been unambiguously established by ¹H NMR and in part by ¹³C NMR, and circular dichroism.     

Inhibition of 5′-nucleotidase activity after growth of Dictyostelium discoideum

The specific activity of 5′-nucleotidase activity in cell-free extracts of Dictyostelium discoideum at both exponential and stationary growth phases was determined. The 5′-nucleotidase activity of both membrane and soluble fractions was determined. The results show that at exponential growth more activity is found in the soluble fraction. Furthermore, the results show that stationary phase cells contain about 10-fold less activity than cells at exponential growth. To determine if stationary phase cells contained an inhibitor of 5′-nucleotidase, purified membranes were incubated with a high speed supernatant (S-100) prepared from cells at this stage. The results showed not only a time and concentration dependent loss of membrane bound activity, but also that most of the lost activity could be recovered in a soluble form. This result suggested that the 5′-nucleotidase was being released by a factor in the S-100. Additional studies showed inactivation of the releasing factor by a protease and further, that this inactivation could be prevented by serine protease inhibitors. The specificity of releasing factor with respect to two other membrane bound activities was determined. The results indicated no loss of either 3′5′-cyclic phosphodiesterase or adenylate cyclase. In addition, the results of a comparison of the activity of the releasing factor at two stages of growth showed similar values at both exponential and stationary growth phase. This latter finding suggests that the loss of 5′-nucleotidase activity at stationary phase is not due to modulation of the releasing factor activity. An alternative mechanism is proposed.