3',5'-Cyclic Guanosine Monophosphate Activates Mitogen-Activated Protein Kinase in Rat Pinealocytes

The role of 3',5'-cyclic guanosine monophosphate (cGMP) in the activation of mitogen-activated protein kinases (MAPKs) was investigated in rat pinealocytes. Treatment with dibutyryl cGMP (DBcGMP) dose-dependently increased the phosphorylation of both p44 and p42 isoforms of MAPK. This effect of DBcGMP was abolished by PD98059 (a MAPK kinase inhibitor), H7 (a nonspecific protein kinase inhibitor), and KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor]. Elevation of cellular cGMP content by treatment with norepinephrine, zaprinast (a cGMP phosphodiesterase inhibitor), or nitroprusside was effective in activating MAPK. Natriuretic peptides that were effective in elevating cGMP levels in this tissue were also effective in activating MAPK. Our results indicate that, in this neuroendocrine tissue, the cGMP/PKG signaling pathway is an important mechanism used by hormones and neurotransmitters in activating MAPK.     

Demonstration of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Cultured Human Schwann Cells

Abstract: Schwann cell cultures were established from adult human sural nerve biopsies. 2'3'-Cyclic nucleotide 3'-phosphohydrolase (CNPase) activity was estimated in the homogenates of those cells by a sensitive isotope assay using [³H]2',3'-cyclic AMP as substrate. A high level of CNPase activity was observed in cultured Schwann cells, whereas cultured human muscle and skin fibroblasts contained negligible levels of CNPase activity. CNPase of human Schwann cells followed typical enzyme-substrate kinetics, with an apparent K _m of 1.6 m M for 2',3'-cyclic AMP, and the enzyme was stimulated by detergents such as Triton X-100 and deoxycholate. It was inhibited by p -chloromercuricbenzoate and 2'-AMP. These properties are typical of CNPase isolated from adult brain and spinal cord. CNPase can serve as a new biochemical marker of normal cultured human Schwann cells and can be useful in analyzing the properties of cultured Schwann cells from patients with dysschwannian neuropathies.     

Attenuation of Enkephalin Activity in Neuroblastoma × Glioma NG108—15 Hybrid Cells by Phospholipases

The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities. Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component. Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.     

Investigations on Myelination In Vitro: Regulation of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase by Thyroid Hormone in Cultures of Dissociated Brain Cells from Embryonic Mice

Abstract: The direct influence of l -3,3',5-triiodothyronine (T₃) on the development of 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37, CNPase) is demonstrated by using an in vitro culture system of dissociated embryonic mouse brain cells. Serum from a thyroidectomized calf, which contained low levels of T₃ (31 ng/100 ml), and thyroxine, T₄ (<1 μg/ml), was used in the culture medium in place of normal calf serum (T₃, 103 ng/100 ml; T₄, 5.7 μg/ml) to render the culture responsive to exogenously added T₃. The lower levels of enzyme activity observed in the presence of such a deficient medium could be restored to normal values by T₃ supplementation. Half-maximal effect was obtained with 2.5 ± 10⁻⁹ m -T₃. Three days of hormone treatment resulted in the maximal stimulation of CNPase. T₄ was less effective in inducing CNPase activity and the inactive analog of the hormone, reverse T₃ (3,3',5'-T₃) was ineffective. The morphological appearance of the cells was characterized by deformed (smaller size and less in number) reaggregates in the cultures, lacking hormone.     

Guanosine 3',5'-monophosphate and the action of alkylating agents.

The intracellular level of guanosine 3',5'-monophosphate (cGMP) has been measured in Walker carcinoma cells in tissue culture after treatment with various alkylating agents. At concentrations which caused a rise in the level of adenosine 3',5'-monophosphate (cAMP) chlorambucil and 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954) produced only a small (35%) elevation of cGMP, while merophan had no such effect. This suggests that any effect of cAMP will not be outweighed by an equivalent rise in cGMP. Sepcific cytosolic binding of cGMP decreased with increasing resistance of Walker cells to alkylating agents, while the dissociation constant, KD, for binding increased. This was also observed with cAMP binding which suggests that the same protein in responsible for binding both nucleotides.     

Differences in cell wall polysaccharides of several species of Eupenicillium

Abstract A β-(1–5)-galactofuran was isolated and characterized from fraction F1S (alkali- and water-soluble) of the cell wall of most of the species of Eupenicillium . In E. cryptum, E. euglaucum and E. nepalense the galactan contained galactofuranose with different linkages in addition to β-(1–5). Fraction F1I (alkali-soluble, water-insoluble) was an α-glucan in certain species while in other it was a =gb-glucan. Xylose was detected in some species in F1I or in F3 (alkali-soluble at 70°C). The most abundant fraction (F4), resistant to the alkali treatment, was a β-glucan-chitin complex. Excepting this component, the β-(1–5)-galactofuran was the polysaccharide which appeared more frequently in the cell wall of species of Eupencillium and it may have chemotaxonomic relevance.     

Bakuchalcone,a dihydrofuranochalcone from the seeds of Psoralea corylifolia

A new dihydrofuranochalcone has been identified in seeds of Psoralea corylifolia and its structure confirmed by synthesis.     

Sugar Composition of Cell Wall Polysaccharides of Suspension-cultured Tobacco Cells

Neutral sugar composition of cell walls of suspension-cultured tobacco cells was examined with the advance of culture age by an anion-exchange chromatography. Isolated cell walls gave on hydrolysis the following sugars: 2% of l-rhamnose, 6% of d-mannose, 26% of l-arabinose, 13% of d-galactose, 8% of d-xylose and 47% of d-glucose as neutral sugars. Little changes in composition of cell wall polysaccharides were recognized with the advance of culture age. Sugar composition of the extra-cellular polysaccharides was similar to that of hemicellulose fraction from cell walls. Pectinic acid gave on hydrolysis 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, d-galacturonic acid and its oligosaccharides.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-Dicarboxycyclopropyl)glycine Binding in Rat Brain

Abstract: [(2S,2′R,3′R)-2-(2′,3′-[³H]Dicarboxycyclopropyl)glycine ([³H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K_D value of 180 ± 33 nM and a B_max of 780 ± 70 fmol/mg of protein. The nonspecific binding, measured using 100 µM LY354740, was <30%. NMDA, AMPA, kainate, l (?)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [³H]DCG IV binding up to 1 mM. However, several compounds inhibited [³H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1′S,2′S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1′S,2′S)-2-methyl-2-(2-carboxycyclopropyl)glycine > l -glutamate = ibotenate > quisqualate > (RS)-α-methyl-4-phosphonophenylglycine = l (+)-2-amino-3-phosphonopropionic acid > (S)-α-methyl-4-carboxyphenylglycine > (2S)-α-ethylglutamic acid > l (+)-2-amino-4-phosphonobutyric acid. N-Acetyl-l -aspartyl-l -glutamic acid inhibited the binding in a biphasic manner with an IC₅₀ of 0.2 µM for the high-affinity component. The binding was also affected by GTPγS, reducing agents, and CdCl₂. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPγS show that [³H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Quantitative determination of the conformation of cyclic 3',5'-adenosine monophosphate in solution using lanthanide ions as nuclear magnetic resonance probes

The conformation of cyclic 3′,5′-adenosine monophosphate in deuterium oxide has been determined at pH 2.0 and pH 5.5, using lanthanide ions as paramagnetic nuclear magnetic resonance probes.The lanthanide ion-induced shifts in the nuclear magnetic resonance energy for a given nucleus are dependent on the geometric position of that nucleus relative to the bound lanthanide ion. As expected, these shifts are pseudocontact in origin and are consistent with axial symmetry. Analysis of the concentration dependence of the shift shows that the lanthanide ion is bound to the phosphate entity giving a 1:1 complex. Further, base stacking and other intermolecular interactions are negligible.To confirm the conformation, which is found from a computer search with the above shift data, we have measured the changes in relaxation times, T₁ and T₂, induced by binding of Gd³⁺. The geometric dependence of these relaxation effects is different from that of shifts, being dependent only on distance. The agreement of these data with the computer “shift” conformation is satisfactory.Some ³¹P nuclear magnetic resonance experiments were done to confirm the metal co-ordination position although, here, there are contact contributions to both shift and relaxation.The computer program finds the conformations that have the correct geometry to account for the shift data, by searching all possible conformations. Non-bond rotations were used as a method of changing the pucker of the phosphate and ribose rings, the position of the base being defined by a single bond rotation. The nuclear magnetic resonance data and minimum van der Waals' distances were used as “active filters” in the computer search.At both values of the pH we have found closely related families of solutions, with the pucker of the phosphate and ribose rings roughly similar to those in an approximate X-ray study of cyclic AMP. The orientation of the base varies with pH.     

Inhibition of ADP-ribosylation of histone by diadenosine 5', 5"' -p (1), p(4)-tetraphosphate

Diadenosine 5′, 5?-p¹, p⁴-tetraphosphate (Ap4A) strongly inhibited ADP-ribosylation reaction of histone by purified bovine thymus poly(ADP-ribose) polymerase. This compound showed a relatively weak inhibitory effect on Mg²⁺-dependent, enzyme-bound poly(ADP-ribose) synthesis. Among various adenine nucleotides tested, including several diadenosine nucleotides with varying phosphate chain length, Ap4A was the most effective inhibitor of the histone-modification reaction. Ap5A and Ap6A showed slightly lower inhibitory effect than Ap4A. Kinetic analysis of the inhibitor (Ap4A) with varying concentration of substrate (NAD⁺) revealed that this compound is a “mixed type inhibitor”, with a Ki value of 5.1 μM.     

Synthesis of (3E, 5Z)-3,5-Dodecadienylacetate,the Sex Pheromone of Phtheochroa cranaodes (Lepidoptera: Tortricidae)

(3E, 5Z)-3,5-Dodecadienyl acetate, the female sex pheromone of Phtheochroa cranaodes, was regio and stereo-selectively synthesized from 1-octyne and (E)-4-bromo-3-buten-1-ol by using Pd(PPh₃)₄, CuI and piperidine to afford the enyne (5). Further elaboration afforded the target pheromone. The synthetic pheromone was identified with the natural product by its MS and IR, data GLC retention time and biological activity.     

Ciglitazone reverses cAMP-induced post-insulin receptor resistance in rat adipocytes in vitro

Ciglitazone (cig), a thiazolidine-dione, lowers glucose and insulin levels in animal models of diabetes type II but not in controls. Since catecholamines given to rat adipocytes in vitro induce insulin resistance similar to that seen in type II diabetes in vivo, we measured the effect of cig on mono-A14-[125I]insulin binding and 3-O-methyl-D-glucose transport (GT) in isolated rat adipocytes treated with isoprenaline (iso, 10 microM). Cig (less than or equal to 5 microM) reversed (ED50 10 nM) the inhibitory effect of iso on insulin stimulation of GT. It had no effect on either basal or insulin stimulated GT. Furthermore, cig did not influence insulin binding either in the presence or absence of iso, which indicates that cig acts only on a post-insulin receptor level. Cig also reversed the inhibition of GT by both forskolin, a cyclase activator and RO20-1724, an imidazolidine phosphodiesterase inhibitor but not that of db-cAMP. It thus seems that cig does not act within the cAMP system but only neutralizes its inhibitory effect on the insulin stimulation of GT.     

Production and purification of a recombinant human 14 kDa β-galactoside-binding lectin

The cDNA for a 14 kDa human β-galactoside-binding lectin was inserted into a plasmid carrying a taq promoter, and the lectin protein was expressed in E. coli cells. The recombinant lectin was extracted from the cells and purified to apparent homogeneity by a single-step chromatography on an asialofetuin-agarose column. Subunit molecular mass (14 kDa), hemagglutinating activity and antigenicity were indistinguishable from those of the human placental lectin. Though the N-terminal of the placental lectin is blocked with an acetyl group, the recombinant lectin was found to have a free amino group. However, the N-terminal amino acid sequences were identical. The recombinant lectin was considered to have the same three-dimensional structure as the placental lectin.     

Chemical and structural similarities in wall polysaccharides of some Penicillium, Eupenicillium and Aspergillus species

Various fractions were extracted from cell-wall material of Eupenicillium crustaceum, Penicillium brevi-compactum, P. decumbens, Aspergillus flavipes and A. ochraceus. The most characteristic fractions, which may have chemotaxonomic relevance, were F1I, an alpha-(1-3) glucan (alkalisoluble, water-insoluble), which amounted to 16.2-32.5% of the cell-wall material, and F1S (alkali and water-soluble) which represented 2.5-6.2% of the cell-wall material and was identified as a beta-(1-5) galactan. 13C-NMR spectra of the F1S fractions showed the same pattern for all the fungal species, characteristic of beta-(1-5) linked galactofuranose.     

3',5' Cyclic nucleotide phosphodiesterase from human platelets: effect of heat upon the multiple forms and their interconversion

A 33,000 g supernatant from human platelets showed a biphasic heat inactivation curve at 45, 50 and 55 degrees C of the cAMP and cGMP phosphodiesterase. This could suggest the presence of two differently heat sensitive phosphodiesterases. However, a preparation heated for 30 min at 55 degrees C, where only the apparently thermostable form of the enzyme remained, still displayed the same characteristics as the starting material, i.e. two apparent Km values for cAMP, a cAMP specific activity lower at low protein concentration (less than 50 micrograms/ml) than at high protein concentration(greater than 100 micrograms/ml), and three peaks of activity upon linear sucrose density gradient. Moreover, a biphasic inactivation curve was again observed after a second heat treatment. These results demonstrated that the heat effect is not a simple protein denaturation of one of two independent species. A study at different temperatures of the profile of the cAMP phosphodiesterase upon sucrose gradient demonstrated that the dissociated form was predominant at high temperature whereas lower temperature favored the associated form. During heat treatment, the dissociated form is at first denatured and this leads to a shift in the equilibrium between the associated and dissociated forms of the phosphodiesterase in favor of the dissociated form. From the overall results, one can draw a model for phosphodiesterase regulation by dissociation-reassociation.     

维生素D_3联合5-氟尿嘧啶对人食管癌Eca-109细胞移植瘤VDR表达的影响

探讨维生素D3、5-氟尿嘧啶单独与联合使用对人食管癌Eca-109细胞移植瘤维生素D受体(vitamin Dreceptor,VDR)的作用.随机分为对照组(C)、预处理组(PT)、维生素D3组(V)、5-氟尿嘧啶组(F)、预处理+5-氟尿嘧啶组(PT+F)、维生素D3+5-氟尿嘧啶组(V+F).体外培养人食管癌Eca-109细胞,BALB/c裸鼠皮下荷瘤,2.5μg/kg1,25-(OH)2维生素D3、25 mg/kg 5-氟尿嘧啶单独与联合腹腔注射,观察瘤体生长情况,逆转录聚合酶链反应(RT-PCR)和蛋白质印迹技术(Western blot)检测裸鼠瘤体组织VDR mRNA与蛋白的表达.研究发现1,25-(OH)2维生素D3、5-氟尿嘧啶均能抑制裸鼠移植瘤的生长,PT、V、F、PT+F、V+F组瘤体体积与C组比较差异有统计学意义(P<0.05);RT-PCR与Western blot结果显示经1,25-(OH)2维生素D3单独与联合5-氟尿嘧啶使用后瘤体组织中VDR mRNA和蛋白表达升高,且联合用药更为显著(P<0.05).结果表明1,25-(OH)2维生素D3、5-氟尿嘧啶均能抑制人食管癌Eca-1...