A SENSITIVE RADIOIMMUNOASSAY FOR 7S NERVE GROWTH FACTOR ANTIGENS IN SERUM AND TISSUES

A radioimmunoassay was developed which can measure accurately concentrations of mouse 7S nerve growth factor antigens (NGFA) as low as 3·0 ng/ml in serum or tissue homogenates. Extremely large amounts of presumed nerve growth factor were found in the submaxillary gland; but considerable quantities were also present in mouse serum, kidney, adrenal gland and vas deferens. Heart, spleen, liver and muscle contained less of the presumed nerve growth factor, and only small amounts were recovered from brain. Rat adrenal gland and serum from rats, guinea pigs and man contained much less immunologically reactive material. The level of presumed nerve growth factor in the mouse heart was highest at birth and decreased slowly during maturation. In the mouse submaxillary gland the content of presumed nerve growth factor increased rapidly after 2 weeks of postnatal age, with higher levels found in male animals. Destruction with 6-hydroxydopamine of the sympathetic nerves in the hearts of newborn or adult mice did not significantly alter the amount of presumed nerve growth factor recovered in the heart.     

Protein kinase C is involved in adrenergic stimulation of pineal cGMP accumulation

The amounts of cAMP and cGMP in the rat pinealocyte are regulated by norepinephrine acting through synergistic dual receptor mechanisms involving alpha 1- and beta-adrenoceptors (Vanecek, J., Sugden, D., Weller, J.L., and Klein, D.C. (1985) Endocrinology 116, 2167-2173; Sugden, L., Sugden, D., and Klein, D.C. (1986) J. Biol. Chem. 261, 11608-11612). Based on the available evidence, it appears that Ca2+-phospholipid-dependent protein kinase is involved in the alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cAMP, but not in the stimulation of cGMP (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W.B. (1985) Nature 314, 359-361). In the present study the role of protein kinase C in the adrenergic stimulation of cGMP was reinvestigated, with the purpose of determining whether protein kinase C activators would potentiate the effects of beta-adrenergic agonists on cGMP if cells were also treated with agents known to elevate intracellular free Ca2+. The protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate (PMA) markedly elevated the cGMP content of beta-adrenergically stimulated pinealocytes which had also been treated with 1 microM A23187, 15 mM K+, or 1 microM ouabain. The effects of A23187 were blocked by EGTA and those of K+ were blocked by nifedipine, establishing the involvement of Ca2+. The stimulatory effects of PMA on cGMP accumulation were mimicked by other protein kinase C activators. PMA also stimulated cGMP accumulation in cells treated with cholera toxin (1 microgram/ml) and A23187 (1 microM), but not in cells treated only with cholera toxin. These results suggest that protein kinase C, which is activated in the pinealocyte by the alpha-adrenergic agonist phenylephrine, is probably involved in the adrenergic regulation of cGMP accumulation at a step distal to receptor activation.     

Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain.

The effects of cAMP-dependent protein kinase (cAMP-PK) phosphorylation on the degradation of the microtubule-associated protein tau by calpain were studied. Purified bovine brain tau that had been phosphorylated by cAMP-PK had a slower migration pattern on sodium dodecyl sulfate-polyacrylamide gels and a more acidic, less heterogeneous pattern on two-dimensional, nonequilibrium pH gradient electrophoresis (NEPHGE) gels compared with untreated tau. Phosphorylation of tau by cAMP-PK significantly inhibited its proteolysis by calpain compared with untreated tau. To our knowledge this is the first demonstration that phosphorylation of tau by a specific kinase results in increased resistance to hydrolysis by calpain. Tau dephosphorylated by alkaline phosphatase migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gels and also showed an altered two-dimensional NEPHGE pattern. Dephosphorylation of tau had no effect on its susceptibility to calpain proteolysis, indicating that regulation of the susceptibility to calpain hydrolysis is due to the phosphorylation of a specific site(s). These results suggest a role for phosphorylation in regulating the degradation of tau. Abnormal phosphorylation could result in a protease-resistant tau population which may contribute to the formation of paired helical filaments in Alzheimer's disease.     

Magnetic circular dichroism and electron paramagnetic resonance studies of hydrogenases I and II from Clostridium pasteurianum

The two iron-only hydrogenases (I and II) from Clostridium pasteurianum have been investigated by variable temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. Samples were studied both reduced with dithionite under an atmosphere of H2 and after oxidation with thionine. The results are consistent with four and two [4Fe-4S]1+,2+ (F)-clusters in hydrogenases I and II, respectively. All four F-clusters are reduced and paramagnetic in reduced hydrogenase I, with up to one exhibiting an S = 3/2 ground state and the remainder having conventional S = 1/2 ground states. Both F-clusters have S = 1/2 ground states in reduced hydrogenase II; however, one appears to be only partially reduced under the conditions used for reduction. MCD studies of the oxidized enzymes show no temperature-dependent features in the visible region which can be attributed to the EPR-active S = 1/2 hydrogen-activating cluster, suggesting predominantly oxygen and nitrogen coordination for the iron atoms of this center. However, temperature-dependent MCD transitions arising from a hitherto undetected S greater than 1/2 Fe-S clusters are apparent in both oxidized hydrogenases. Detailed EPR studies of oxidized hydrogenase I revealed resonances from an S = 3/2 species, however, spin quantitation reveals this to be a trace component that is unlikely to be responsible for the observed low temperature MCD spectrum. The nature and origin of these S greater than 1/2 Fe-S clusters are discussed in light of the available spectroscopic data for these and other iron-only hydrogenases.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.