Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.     

Growth stimulation by PGE2 and EGF activates cyclic AMP-dependent and -independent pathways in primary cultures of mouse mammary epithelial cells

Mammary epithelial cells from virgin Balb/c mice were isolated by collagenase digestion and cultured within collagen gels in serum-free basal medium containing insulin (10 micrograms/ml). Previous work has shown that linoleate or its metabolite, prostaglandin E2 (PGE2), stimulate the growth of these cells only in the presence of a growth stimulant such as epidermal growth factor (EGF). Since PGE2 can stimulate cyclic AMP (cAMP) production, the role of cAMP in linoleate and EGF-stimulated growth was examined. The cAMP phosphodiesterase inhibitor, IBMX (0.1 mM), was found to augment growth when cells were cultured in the presence of both EGF and linoleate or PGE2, but not either factor alone. These results indicated that EGF does not stimulate proliferation via cyclic AMP mediated events but could synergize with cAMP events if cAMP levels were elevated by PGE2. When assayed in cells plated on top of collagen-coated culture dishes, cellular cyclic AMP levels were stimulated by PGE2, but only marginally by EGF. Although the stimulation of endogenous cAMP by PGE2 and IBMX was insufficient to stimulate growth in the absence of EGF, exogenous dibutyryl-cAMP (greater than 100 micrograms/ml) was able to do so showing that a sustained, and high level of cAMP (greater than 100 micrograms/ml) could stimulate growth in insulin-containing basal medium. EGF was capable of enhancing the cellular sensitivity to dibutyryl-cAMP but the converse was not observed. cAMP stimulation of growth was dependent upon a superphysiological concentration of insulin (10 micrograms/ml) or a physiological concentration of somatomedin-C. These results indicate that the proliferation of mouse mammary epithelial cells can be stimulated separately or in synergism by cAMP-dependent or -independent events.     

A group contribution method for the estimation of equilibrium constants for biochemical reactions

Using Gibbs Energies of compounds, as well as Gibbs Energy changes and equilibrium constants of biochemical reactions, the contributions of functional groups to the Gibbs Energy (in aqueous solution, temperature 25°C, and pH=7) have been estimated. These contributions allow the estimation of the Gibbs Free Energy and the equilibrium constant of a biochemical reaction, given the structure of the reactants and products.     

Oxidative Damage to DNA and Deoxyribose by β-Lactam Antibiotics in the Presence of Iron and Copper Salts

β-lactam antibiotics in the presence of certain metal ions damage deoxyribose and DNA with the release of thiobarbituric acid-reactive material. This damage can be substantially prevented by catalase, metal chelators and some scavengers of the hydroxyl radical. Ferric salts in the presence of certain β-lactam antibiotics were effective in degrading deoxyribose but they did not appear to damage DNA. In contrast copper salts and p-lactam antibiotics were extremely effective in damaging both DNA and deoxyribose.     

An improved method for primary culture of ovarian androgen-producing cells in serum-free medium: Effect of lipoproteins,insulin, and insulinlike growth factor-I

Summary Although luteinizing hormone (LH) alone stimulates ovarian interstitial cells cultured in serum-free medium to synthesize large amounts of androgens, there seem to be additional factors in vivo that modulate the time course and magnitude of the cellular responses to LH. In an attempt to develop a more nearly physiologic cell culture model, lipoproteins, insulin, and insulinlike growth factor-I (IGF-I) were added to the serum-free medium. The effects of these modifications on androgen biosynthesis by dispersed cells from ovaries of hypophysectomized immature rats cultured in 96-well tissue culture plates were examined. A saturating dose of LH stimulated a 25-fold increase in androsterone synthesis at 2 d, which decreased at 4 and 6 d. Addition of human high density (hHDL) or human low density lipoprotein (hLDL) caused a 2.5-fold increase in LH-stimulated androsterone synthesis. Cells were approximately twice as sensitive to hHDL (ED₅₀=5.5±0.5 μg cholesterol/ml) compared to hLDL (ED₅₀=9.1±1.1 μg cholesterol/ml). Surprisingly, rat HDL caused only a 40% increase in LH-stimulated androsterone synthesis. When insulin alone was added to cells cultured with a saturating dose of LH, there was a 2.8-fold increase in androsterone synthesis. Addition of hHDL and insulin together caused a synergistic increase in LH-stimulated androsterone synthesis. In contrast to hHDL, which did not change the time course of LH-stimulated androsterone production, insulin prolonged maximal LH-stimulated androsterone synthesis at 4 and 6 d. Inasmuch as the ED₅₀ for insulin action (1.3±0.1 μg/ml) was supraphysiologic, the effects of IGF-I on LH-stimulated androgen synthesis were examined. IGF-I mimicked all of the effects of insulin, but at a physiologic concentration (ED₅₀=2.5±0.3 ng/ml). Ovarian cells cultured in serum-free medium supplemented with hHDL and insulin or IGF-I exhibit responses that closely approximate the physiologic responses observed in vivo. These results suggest that lipoproteins and IGF-I are important physiologic stimulators of ovarian theca-interstitial cell androgen biosynthesis which, when added to the serum-free medium, make the cellular responses in this in vitro model more nearly approximate the responses in vivo. This research was supported by research center grant HD 12303 from the National Institute of Child Health and Human Development, Bethesda, MD, and USCD Academic Senate grant RM-169M     

Ammonia regulation of phosphate-activated glutaminase displays regional variation and impairment in the brain of aged rats

The regulation of PAG by ammonia in whole brain (Sprague-Dawley) and regional (Fischer-344) synaptosomal preparations from adult and aged animals was assessed. Whole brain synaptosomal preparations from both age groups displayed a significant decrease in PAG activity with increasing ammonium chloride concentrations, however, the aged rats exhibited a significant attenuation in ammonia-induced PAG inhibition. PAG activity measured in synaptosomes prepared from the striatum (STR), temporal cortex (TCX) and hippocampus (HIPP) was also inhibited by ammonium chloride. The STR showed the greatest degree of ammonia-induced PAG inhibition (55%) followed by the HIPP (30–35%) and the TCX (25–30%). This reduction in PAG activity was significantly attenuated in STR from aged rats at ammonium chloride concentrations greater than 50 M and in the TCX, PAG activity was significantly attenuated in the aged rats at ammonia concentrations of 0.5 and 1.0 mM. Ammonia regulation of PAG activity in the HIPP appeared to be unaffected by age. Ammonium chloride concentrations up to 5 mM had no effect on GLU release from cortical slices, although GLN efflux was significantly enhanced. These findings suggest that isozymes of PAG may exist in different brain regions based on their differential sensitivity to ammonia. The attenuation of ammonia-induced PAG inhibition seen in aged rats may have deleterious effects in the aged brain.Abbreviations PAG phosphate-activated glutaminase: L-glutamine amidohydrolase; EC 3.5.1.2 - STR striatum - TCX temporal cortex - HIPP hippocampus     

Selective isolation of bacteria with dipeptidyl aminopeptidase type I activity from the sheep rumen

Five-hundred-and-six fresh isolates of rumen bacteria were tested for their ability to hydrolyse the synthetic substrate for dipeptidyl aminopeptidase type I, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), using a gel overlay technique. Twelve positive isolates were small Gram-negative rods which resembled Bacteroides ruminicola in their biochemical and morphological properties. SDS-PAGE of whole cell extracts indicated that two were similar to B. ruminicola strain B14, six resembled B. ruminicola strain M384, and four were similar to B. ruminicola GA33. All hydrolysed GlyArg-MNA, Ala2 and Ala5, and showed no activity against Leu-MNA. Ala3 and Ala2, but no Ala4, was produced from Ala5. The different groups had different, distinctive activity profiles. The two remaining positive isolates were Lactobacillus spp. with an exceptionally high Leu-MNA activity. It was concluded that, although different strains may only be distantly related, B. ruminicola forms the most important group of bacteria in the rumen to possess a dipeptidyl aminopeptidase type I activity.     

Somatic embryogenesis and plant regeneration from zygotic embryo explants in mexican weeping bamboo,Otatea acuminata aztecorum

An efficient protocol has been developed for the in vitro propagation of Mexican Weeping Bamboo through somatic embryogenesis from zygotic embryo explants. Mature seeds and excised embryos were cultured in the light or in the dark on both Murashige and Skoog's and Gamborg's B5 basal media with various supplements. Optimal somatic embryogenesis and plant regeneration were obtained by culture in the dark on Murashige and Skoog's basal medium supplemented with 3 mg/1 2,4-dichlorophenoxyacetic acid, 0.5 mg/1 6-benzylaminopurine and 2.0% sucrose. More than 95% of the germinating somatic embryos developed shoots and roots, and were transferred to soil with 85% success.     

Nucleoside triphosphates are required to open the CFTR chloride channel.

The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.     

Isolation of a yeast mutant deficient in pyruvate carboxylase activity

To improve our understanding of the catalytic mechanism and regulatory properties of pyruvate carboxylase (EC 6.4.1.1), an important biotin-dependent enzyme, we have sought to isolate mutants in Saccharomyces cerevisiae which are defective in pyruvate carboxylase activity. One mutant was isolated which was unable to grow on glucose minimal medium unless supplemented with aspartate. Although the enzyme had only 25% of the wild type pyruvate carboxylase activity, Western analysis and RNase protection analysis demonstrated that the mutant gene was expressed at approximately 70% of the wild type level. On the basis of genetic crosses and complementation tests, we have attributed the defect to mutations in the PYC gene encoding pyruvate carboxylase.