Neutral Amino Acid Transport in Astrocytes: Characterization of Na+-Dependent and Na+-Independent Components of α-Aminoisobutyric Acid Uptake

Neutral amino acid transport is largely unexplored in astrocytes, although a role for these cells in blood-brain barrier function is suggested by their close apposition to cerebrovascular endothelium. This study examined the uptake into mouse astrocyte cultures of alpha-aminoisobutyric acid (AIB), a synthetic model substrate for Na+-dependent system A transport. Na+-dependent uptake of AIB was characteristic of system A in its pH sensitivity, kinetic properties, regulatory control, and pattern of analog inhibition. The rate of system A transport declined markedly with increasing age of the astrocyte cultures. There was an unexpectedly active Na+-independent component of AIB uptake that declined less markedly than system A transport as culture age increased. Although the saturability of the Na+-independent component and its pattern of analog inhibition were consistent with system L transport, the following properties deviated: (1) virtually complete inhibition of Na+-independent AIB uptake by characteristic L system substrates, suggesting unusually high affinity of the transporter; (2) apparent absence of trans-stimulation of AIB influx; (3) unusually concentrative uptake at steady state (the estimated distribution ratio for 0.2 mM AIB was 55); and (4) susceptibility to inhibition by N-ethylmaleimide. Direct study of the uptake of system L substrates in astrocytes is needed to confirm the present indications of high affinity and concentrative Na+-independent transport.     

Neuropeptide-γ: A Peptide Isolated from Rabbit Intestine that Is Derived from γ-Preprotachykinin

The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val- Gly-Leu - Met.NH2. This amino acid sequence represents residues (72-92) of gamma-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-gamma, lacks residues (3-17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of beta- and gamma-preprotachykinins in the rabbit gut are different.     

Structural Features of Human Monoamine Oxidase A Elucidated from cDNA and Peptide Sequences

Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neuropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO-A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C-terminal region. The presence of features characteristic of the ADP-binding fold suggests that the N-terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Induction by antimycin A of cyanide-resistant respiration in heterotrophic Euglena gracilis: Effects of growth,respiration and protein biosynthesis

The addition of antimycin A during the logarithmic phase of growth of heterotrophic Euglena gracilis cultures (in lactate or glucose medium) was immediately followed by decreased respiration and a cessation of grwoth. Induced cyanideresistent respiration appeared 5 h after the addition of the inhibitor then the cells started to grow again and could be cultured in the presence of antimycin A. Thus the cells exhibited a cyanide-and antimycin-resistant respiration which was, in addition, sensitive to salicylhydroxamic acid and propylgallate. Antimycin-adapted Euglena and control cells were compared for their biomass production and protein synthesis. The difference in growth yield between control and antimycin-adapted cells was not as high as would be expected if only the first phosphorylation site of the normal respiratory chain was active in the presence of antimycin A. Furthermore, the ability to incorporate labelled valine into proteins, under resting-cell conditions, was not changed. Strong correlations were established between the effects of respiratory effectors on O₂ consumption and valine incorporation. These results suggest that sufficient energy for protein synthesis and growth is provided by the operation of the cyanide-resistant respiratory pathway in antimycin-adapted Euglena.Abbreviations DNP dinitrophenol - PG propylgallate - SHAM salicylhydroxamic acid     

Characterization of a delayed rectifier K+ channel in NG108-15 neuroblastomaX glioma cells: Gating kinetics and the effects of enrichment of membrane phospholipids with arachidonic acid

Summary The calcium sensitivity of exocytosis from electroper-meabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electropermeabilized cells are exposed to Ca²⁺ levels much in excess of 50 m. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca²⁺ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.Deceased     

Deglycosylation of a lectin intermediate during assembly of ConA

Summary Metabolic labelling of immature jackbeans (Canavalia ensiformis) has been used in a pulse-chase study to determine changes in the glycosylation pattern of polypeptides during the assembly of Concanavalin A. In an analysis that allowed the identification of 7 intermediates, only the first precursor form of the lectin was labelled with D-[U-¹⁴C]-glucosamine. These results indicate that processing of the lectin involves a novel deglycosylation event in which an N-linked oligosaccharide is removed from a protein in the absence of proteolysis.Abbreviations endo H endo -N-acetylglucosaminidase H - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - ConA Concanavalin A     

水稻原生质体产生细胞团的冰冻保存和冻后再生植株形成

水稻(Oryza sativa L.)原生质体产生的细胞团加上10-20％的二甲亚枫(DMSO)和10-20％的蔗糖,置于液氮中保存。冻后细胞生存率达到对照的40-50％。存活的细胞在附加2×10~(-5)mol/l 2,4-D 的Linsmier-Skoog(Ls)固体培养基上再生长,然后将形成的愈伤组织块转到附加10~(-6)mol/l NAA,4×10~(-6)mol/l 激动素和10~(-6)mol/l 2 IP 及8％的蔗糖的 LS培养基上分化出芽并形成植株。     

Relationship of cellular oncogene expression to inhibition of growth and induction of differentiation of Daudi cells by interferons or TPA

Human alpha or beta interferons inhibit the proliferation of Daudi Burkitt lymphoma cells and induce the differentiation of these cells towards a mature plasma cell phenotype. Similar responses are seen when Daudi cells are treated with the phorbol ester, TPA. Both interferons and TPA down-regulate expression of the c-myc oncogene in these cells. Although TPA can mimic the effect of interferon on cell differentiation, it does not induce 2'5' oligoadenylate synthetase or the interferon-sensitive mRNAs, 6-16 or 9-27. Thus chronic stimulation of protein kinase C by TPA cannot mimic all of the effects of interferon treatment on gene expression. Inhibition of ADP-ribosyl transferase activity by 3-methoxybenzamide impairs interferon- or TPA-induced differentiation of Daudi cells. This agent induces a higher level of c-myc mRNA in the cells and stimulates the incorporation of [3H]thymidine into DNA; although these effects are partially counteracted by interferon or TPA treatment, the elevated expression of the c-myc gene may be sufficient to prevent terminal differentiation and allow cell proliferation to continue.     

Regulation of nicotinic acetylcholine receptors by protein phosphorylation

Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed.