The translational stop signal: Codon with a context, or extended factor recognition element?

Wide ranging studies of the readthrough of translational stop codons within the last 25 years have suggested that the stop codon might be only part of the molecular signature for recognition of the termination signal. Such studies do not distinguish between effects on suppression and effects on termination, and so we have used a number of different approaches to deduce whether the stop signal is a codon with a context or an extended factor recognition element. A data base of natural termination sites from a wide range of organisms (148 organisms, 40000 sequences) shows a very marked bias in the bases surrounding the stop codon in the genes for all organisms examined, with the most dramatic bias in the base following the codon (+4). The nature of this base determines the efficiency of the stop signal in vivo, and in Escherichia coli this is reinforced by overexpressing the stimulatory factor, release factor-3. Strong signals, defined by their high relative rates of selecting the decoding release factors, are enhanced whereas weak signals respond relatively poorly. Site-directed cross-linking from the +1, and bases up to +6 but not beyond make close contact with the bacterial release factor-2. The translational stop signal is deduced to be an extended factor recognition sequence with a core element, rather than simply a factor recognition triplet codon influenced by context.     

Presynaptic proteins involved in exocytosis inDrosophila melanogaster: A genetic analysis

Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal. This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane. Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human. Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking. This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion. The hypothesis that these mechanisms function at the synapse relies largely uponin vitro evidence. Recently, genetic approaches in mice, C.elegans and the fruitfly,Drosophila melanagaster, have been used to dissect thein vivo function of numerous proteins involved in synaptic transmission. This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release inDrosophila. In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, makingDrosophila an ideal model system to further unravel the intricacies of synaptic transmission.     

Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Analysis of Its Action Site Using Sepiapterin

Abstract: Recently, we reported that 6 R - l - erythro -tetrahydrobiopterin (6 R -BH₄), a natural cofactor for hydroxylases of tyrosine and tryptophan, has a monoamine-releasing action independent of its cofactor activity. Here we attempted to determine whether 6 R -BH₄ acts inside the cell or from the outside of the cell by using brain microdialysis in the rat striatum. For this purpose, sepiapterin, an immediate precursor of 6 R -BH₄ in the salvage pathway, was used to selectively increase the intracellular 6 R -BH₄ levels. Dialytic perfusion of sepiapterin increased tissue levels of reduced biopterin (mainly 6 R -BH₄) but not the extracellular levels. Administration of sepiapterin increased the extracellular levels of 3,4-dihydroxyphenylalanine (DOPA) (an index of in vivo tyrosine hydroxylase activity) and of dopamine (DA) (an index of in vivo DA release). Either of the increases was eliminated after pretreatment with a tyrosine hydroxylase inhibitor α-methyl- p -tyrosine. Administration of 6 R -BH₄ increased extracellular levels of reduced biopterin, DOPA, and DA. After pretreatment with α-methyl- p -tyrosine, the increase in DOPA levels was abolished, but most of the increase in DA levels persisted. The increase in DA levels also persisted after pretreatment with nitric oxide synthase inhibitors. These data demonstrate that 6 R -BH₄ stimulates DA release directly, independent of its cofactor action for tyrosine hydroxylase and nitric oxide synthase, by acting from the outside of neurons.     

Fenfluramine Releases Serotonin from Human Brain Nerve Endings by a Dual Mechanism

Abstract: Fenfluramine is the most widely used anorexigenic drug in humans. In animal experiments d -fenfluramine has been shown to act as a potent releaser of brain serotonin [5-hydroxytryptamine (5-HT)]. Here we have investigated the effects of d -fenfluramine on the release of [³H]5-HT from isolated nerve endings of human neocortex. The drug elicited release of unmetabolized [³H]5-HT, and this effect was concentration dependent. However, the mechanism of release seems to differ profoundly depending on the concentrations of d -fenfluramine used. At 5 µ M , the release of [³H]5-HT was blocked by the 5-HT transporter inhibitor fluoxetine and was Ca²⁺ independent and insensitive to the human autoreceptor 5-HT_1D agonist sumatriptan. The release of [³H]5-HT elicited by 0.5 µ M d -fenfluramine was similarly blocked by fluoxetine, but it was strongly Ca²⁺ dependent and sensitive to sumatriptan. It is suggested that, at relatively high concentrations, d -fenfluramine largely diffuses into serotonergic terminals and causes release of 5-HT through the 5-HT carrier working in the inside-outside direction; at relatively low concentrations d -fenfluramine enters the terminals through the 5-HT transporter but elicits release of 5-HT by an exocytotic-like mechanism.     

Protein Kinase FA/Glycogen Synthase Kinase-3α After Heparin Potentiation Phosphorylates τ on Sites Abnormally Phosphorylated in Alzheimer''s Disease Brain

In this work, we tested the effect of ion channel blockers and of phorbol ester treatments on [³H]dopamine ([³H]DA) release and neurotensin (NT)-induced facilitation of [³H]DA release from cultures of rat fetal mesencephalic cells. The potassium channel blockers tetraethylammonium and 4-aminopyridine increased basal [³H]DA release and decreased K⁺-evoked [³H]DA release, whereas apamin was without effect. K⁺-evoked [³H]DA release was decreased by ω-conotoxin and nifedipine, totally suppressed by cadmium, and unaffected by amiloride. These results show the differential sensitivity of [³H]DA release to blockade of various ion channels and suggest the involvement of N-type, L-type, and non-L-non-N-type, but not T-type, voltage-sensitive calcium channels in K⁺-evoked release. Phorbol 12-myristate 13-acetate increased both spontaneous and K⁺-evoked [³H]DA release, suggesting a modulatory action of protein kinase C on DA release in this system. Unexpectedly, however, the effects of the phorbol ester were not counteracted by the protein kinase C inhibitors H7, staurosporine, or polymyxin B. NT-induced facilitation of K⁺-evoked [³H]DA release was insensitive to most of the ion channel blockers, except cadmium (64% decrease in NT effect), suggesting that the corresponding potassium' and calcium channels were not involved in the effect of NT on [³H]DA release in this system. The NT effect was totally suppressed by phorbol ester treatments, indicating a possible desensitization of the corresponding transduction mechanisms after protein kinase C activation.     

Chronic Nicotine Administration Differentially Affects Neurotransmitter Release from Rat Striatal Slices

Abstract: The objective of these experiments was to determine whether the chronic administration of nicotine, at a dose regimen that increases the density of nicotine binding sites, alters the nicotine-induced release of [³H]dopamine ([³H]DA), [³H]norepinephrine ([³H]NE), [³H]serotonin ([³H]5-HT), or [³H]acetylcholine ([³H]ACh) from rat striatal slices. For these experiments, rats received subcutaneous injections of either saline or nicotine bitartrate [1.76 mg (3.6 µmol)/kg, dissolved in saline] twice daily for 10 days, and neurotransmitter release was measured following preloading of the tissues with [³H]DA, [³H]NE, [³H]5-HT, or [³H]choline. Chronic nicotine administration did not affect the accumulation of tritium by striatal slices, the basal release of radioactivity, or the 25 mM KCl-evoked release of neurotransmitter. Superfusion of striatal slices with 1, 10, and 100 µM nicotine increased [³H]DA release in a concentration-dependent manner, and release from slices from nicotine-injected animals was significantly (p < 0.05) greater than release from saline-injected controls; release from the former increased to 132, 191, and 172% of release from the controls following superfusion with 1, 10, and 100 µM nicotine, respectively. Similarly, [³H]5-HT release increased in a concentration-related manner following superfusion with nicotine, and release from slices from nicotine-injected rats was significantly (p < 0.05) greater than that from controls. [³H]5-HT release from slices from nicotine-injected rats evoked by superfusion with 1 and 10 µM nicotine increased to 453 and 217%, respectively, of release from slices from saline-injected animals. The nicotine-induced release of [³H]NE from striatal slices was also concentration dependent but was unaffected by chronic nicotine administration. [³H]ACh release from striatal slices could not be detected when samples were superfused with nicotine but was measurable when tissues were incubated with nicotine. The release of [³H]ACh from slices from nicotine-injected rats was significantly (p < 0.05) less than release from controls and decreased to 36, 83, and 77% of control values following incubation with 1, 10, or 100 µM nicotine, respectively. This decreased [³H]ACh release could not be attributed to methodological differences because slices from nicotine-injected rats incubated with nicotine exhibited an increased [³H]DA release, similar to results from superfusion studies. In addition, it is unlikely that the decreased release of [³H]ACh from striatal slices from nicotine-injected rats was secondary to increased DA release because [³H]ACh release from slices from hippocampus, which is not tonically inhibited by DA, also decreased significantly (p < 0.05) in response to nicotine; hippocampal slices from nicotine-injected rats incubated with 1 and 10 µM nicotine decreased to 42 and 70%, respectively, of release from slices from saline-injected animals. Results indicate that the chronic administration of nicotine increases the ability of nicotine to induce the release of [³H]DA and [³H]5-HT and decreases the ability of nicotine to evoke the release of [³H]ACh but does not alter the nicotine-induced release of [³H]NE from brain slices.     

Cyclothiazide Selectively Potentiates AMPA- and Kainate-Induced [3H]Norepinephrine Release from Rat Hippocampal Slices

Abstract: Activation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of ionotropic glutamate receptors has been shown to result in a rapid desensitization of the receptor in the presence of certain agonists. One effect of AMPA receptor desensitization in the hippocampus may be to decrease the efficacy of AMPA receptor agonists at stimulating the release of norepinephrine from noradrenergic terminals. Recently, cyclothiazide was reported to inhibit AMPA receptor desensitization by acting at a distinct site on AMPA receptors. We have examined the effect of cyclothiazide on AMPA- and kainate (KA)-induced norepinephrine release from rat hippocampal slices to determine whether cyclothiazide would increase the efficacy of AMPA-induced [³H]norepinephrine release by inhibiting AMPA receptor desensitization. Cyclothiazide was observed to potentiate markedly both AMPA- and KA-induced [³H]norepinephrine release. This potentiation is selective for AMPA/KA receptors as cyclothiazide did not potentiate N -methyl- d -aspartate-induced [³H]norepinephrine release or release induced by the nonspecific depolarizing agents veratridine and 4-aminopyridine. These results demonstrate that AMPA receptor-mediated modulation of [³H]norepinephrine release from rat brain slices is a useful approach to studying the cyclothiazide modulatory site on the AMPA receptor complex.     

α2-Adrenoceptor-Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [³H]noradrenaline release and in the α₂-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α₂-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on ³H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [³H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca²⁺ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca²⁺ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α₂-adrenergic inhibition of noradrenaline release involves voltage-activated Ca²⁺ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α₂-adrenergic reduction, apparently through a disinhibition of Ca²⁺ channels.     

Polymeric derivatives of plant growth regulators: synthesis and properties

The polymeric formulations of plant growth regulators (PGRs) are high molecular weight systems in which the PGR unit is attached to the polymeric chain by a hydrolysable chemical bond. These polymeric derivatives (esters, ethers, or else) of PGRs are characterised by the ability to release the active compound (PGR) from their solutions (mainly aqueous) in certain conditions. The release of the PGR can be controlled by external factors (pH, temperature, enzymes, solution concentration), and inherent properties of the whole macrosystem chemical structure, such as the type of the hydrolysable bond between PGR unit and the main polymeric chain, the structure of the polymer chain (e.g. molecular weight, level of hydrophilicity, and the content of hydrophobic groups, macromolecular conformation in solution etc.). These controlled (slow) release PGRs display certain advantages over conventional PGR formulations due to their prolonged action, improved efficiency (e.g. wide range of effective concentrations) greater safety to non-target organisms and the applicators. In addition the ability of altering the solubility level and modifying the aplication form is of considerable interest. The biological activity efficiency of polymeric PGRs has been documented and the relation of this efficiency to the PGR unit hydrolytic release ability has been mentioned. Slow release polymeric PGRs are considered to solve certain problems in agriculture.Abbreviations PGR plant growth regulator - C(S)RF controlled (slow) release form - PD polymeric derivative - ACC 1-amino-cyclopropane-1-carboxylic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - BAP N⁶-benzylaminopurine - ABA abscisic acid - GA gibberellin - LMW low molecular weight - HMW high molecular weight     

Enhancement of extractable ethylene at light/dark transition in primary leaves of paclobutrazol-treated Phaseolus vulgaris seedlings

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol], a triazole growth retardant, increased the 1-aminocyclopropane-1-carboxylic acid (ACC) level and resulted in reduced ethylene production, estimated as ethylene release in a closed system or by vacuum-extraction, in the primary leaves of Phaseolus vulgaris L. cv. Juliska seedlings exposed to light. At the light/dark transition, a definite enhancement of the endogenous ethylene level was observed by vacuum-extraction of primary leaves of treated plants and the ethylene deficiency of retardant-treated leaves ceased. The concentration of ACC after the light/dark transition followed the pattern for ethylene, and the increase in ACC content was paralleled by a decrease in malonyl-ACC.
It is concluded that the internal level of ethylene is not necessarily lower in the primary leaves of paclobutrazol-treated bean plants, but under special environmental conditions in vivo it may reach that of the control.