EFFECT OF LIGHT ON AUXIN TRANSPORT AND ELONGATION OF AVENA MESOCOTYL

The present work was undertaken to find if there are relations between light and auxin action on elongation of coleoptilar node and mesocotyl with Avena seedlings. Red light inhibited the elongation of mesocotyl and simultaneously decreased the rate of transport of diffusible auxin through the node. Red light also inhibited the transport of exogenously given IAA through the nodal region. The light inhibition of IAA transport was closely related to the increase of IAA immobilization. As the age proceeds, the ability of IAA immobilization increased with the decrease in the rate of mesocotyl elongation, even if the seedling was grown in complete darkness. The nature of radioactive substances found in the IAA-C¹⁴ treated tissue was examined by paper chromatography. The above results strongly suggested that the increase of IAA immobilization might result in the inhibition of mesocotyl elongation.     

Specificity of Auxin-binding Sites on Maize Coleoptile Membranes as Possible Receptor Sites for Auxin Action

Dissociation coefficients of auxin-binding sites on maize (Zea mays L.) coleoptile membranes were measured, for 48 auxins and related ring compounds, by competitive displacement of ¹⁴C-naphthaleneacetic acid from the binding sites. The sites bind with high affinity several ring compounds with acidic side chains 2 to 4 carbons long, and much more weakly bind neutral ring compounds and phenols related to these active acids, most phenoxyalkylcarboxylic acids, and arylcarboxylic acids except benzoic acid, which scarcely binds, and triiodobenzoic acids, which bind strongly. Specificity of the binding is narrowed in the presence of a low molecular weight “supernatant factor” that occurs in maize and other tissues. Activity of many of the analogs as auxin agonists or antagonists in the cell elongation response was determined with maize coleoptiles. These activities on the whole roughly parallel the affinities of the binding sites for the same compounds, especially affinities measured in the presence of supernatant factor, but there are some quantitative discrepancies, especially among phenoxyalkylcarboxylic acids. In view of several factors that can cause receptor affinity and biological activity values to diverge quantitatively among analogs, the findings appear to support the presumption that the auxin-binding sites may be receptors for auxin action.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

Evidence for Alpha-MSH Binding Sites on Human Scalp Hair Follicles: Preliminary Results

Alpha-MSH, considered an important pigmentation hormone, binds to melanocytes and is thought to stimulate melanogenesis through a cyclic-AMP-dependent mechanism. The binding of alpha-MSH to follicular melanocytes has been investigated in human hair of different colors, ranging from black to blond and senile white. Hairs were plucked, the follicles were cut off, and an alpha-MSH binding assay, using a radiolabeled alpha-MSH analogue, was performed on these bulbs. As controls of each assay, fragments of hairs of the same person were used. The results show a dose-response relationship and the assay seems to be specific for alpha-MSH, because other peptides such as ACTH, beta-LPH and beta-endorphins do not compete for binding sites as alpha-MSH does. These binding sites seem to be present only on melanin synthesizing melanocytes, since the controls and follicles of senile white hair, which do not contain active melanocytes, show negative results. All the assays were performed on raw material, i.e., whole plucked hair follicles. This is the first time that binding sites for alpha-MSH have been demonstrated on human scalp hair follicles. In addition, their presence was found to be associated with active melanin production; their absence was demonstrated on senile white hair follicles.     

Evidence for Functional Activity of Up-Regulated Nicotine Binding Sites in Rat Striatal Synaptosomes

A number of studies have found that the chronic administration of nicotine causes an increase in the density of nicotinic binding sites in the brain, but it is not known whether these additional binding sites are functionally active receptors. In this study, the effects of 1-week administration of the potent nicotinic agonist, (+)-anatoxin-a (96 nmol/day via osmotic minipumps), was assessed on [3H]nicotine binding and [3H]dopamine uptake and release in rat striatal synaptosomes. Chronic (+)-anatoxin-a treatment resulted in a 32% increase in the Bmax of [3H]nicotine binding in anatoxin-treated animals compared to control. There was a 43% increase in the activity of 3 microM nicotine to release [3H]dopamine from synaptosomes of anatoxin-treated animals, but the release induced by 20 mM K+ depolarization was unaffected. There was no effect of chronic (+)-anatoxin-a treatment on the uptake of [3H]dopamine. A strong positive correlation (r = 0.64) was found between the density of [3H]nicotine binding sites and the nicotine-induced stimulation of [3H]dopamine release in individual animals. These results indicate that (+)-anatoxin-a, like nicotine, produces an up-regulation of nicotine binding sites following chronic administration, and that these additional sites are functional receptors capable of mediating the release of dopamine from striatal synaptosomes.     

环糊精葡萄糖基转移酶钙离子结合位点结构与功能分析

通过多重序列比对和晶体结构分析发现,钙离子结合位点CaⅠ和CaⅡ普遍存在于环糊精葡萄糖基转移酶(CGT酶)中,且两个位点处氨基酸残基具有较高的保守性,而钙离子结合位点CaⅢ仅存在于少数CGT酶中．此外,研究发现,钙离子结合位点可能与CGT酶的环化活力、热稳定性和产物特异性密切相关．     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Evidence for a Reduction of Coupling between GABA<Subscript>A</Subscript> Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[³⁵S]TBPS binding to the GABA_A receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [³⁵S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [³⁵S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [³H]flunitrazepam binding to benzodiazepine site of recombinant α1β2γ2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [³⁵S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABA_A receptors. Special issue dedicated to Simo S. Oja     

Evidence for the Existence of Imidazoline-Specific Binding Sites in Synaptosomal Plasma Membranes of the Bovine Brainstem

Abstract: Nonadrenergic imidazoline-specific binding sites were characterized pharmacologically in crude cerebral membrane preparations, but little is known about their subcellular localization in neurons. As in the brain-stem these sites are involved in cardiovascular regulation and peripherally imidazolines modulate neurotransmitter release, we tried to determine a possible (pre)synaptic localization in brainstem. We found a specific enrichment in (entire) synaptosome, purified synaptosomal plasma membrane (37 fmol/mg), and mitochondrial (83 fmol/mg) fractions as compared with other membrane fractions (3–8 fmol/mg). Synaptosomes appeared to be free of postsynaptic structures, and purified synaptosomal plasma membranes were devoid of mitochondrial material, as determined by electron microscopy and by comparison with the distribution of marker enzymes such as monoamine oxidase. These results show for the first time that these extramitochondrial imidazoline-specific sites are neuronal and are located on presynaptic terminals. We found high affinities for unlabeled p -iodoclonidine (subnanomolar), clonidine (0.2 n M ), and efaroxan (11 n M ), but idazoxan did not compete significantly for the p -[¹²⁵I]iodoclonidine binding in these membranes. Therefore, these sites can be classified as I₁ imidazoline receptors. In summary, we describe for the first time that high-affinity I₁ receptors of the bovine brainstem are located on (pre)synaptic membranes.     

Studies in the Physiological Action of 2, 2-Dichloropropionic Acid: I.: MECHANISMS CONTROLLING THE INHIBITION OF ROOT ELONGATION

The inter-relationships between time and concentration and thedegree of inhibition of root elongation have been examined forSorghum vulgare, Zea mays, Helianthus annuus, and Pisum sativum.For all species the inhibitory effect is cumulative but thereis a tenfold difference in the concentration required to halvethe elongation of the most sensitive (S. vulgare) and most resistantspecies (P. sativum). From a comparison of the growth of intactsroots and isolated segments, together with estimates of cellnumber, it has been established that the primary effect is tointerfere with meristematic activity in the root tip, wherethe mitotic cycle is arrested at prophase. Using 2, 2-dichloropropionic acid, containing chlorine-36, thecourses of uptake by both intact roots and isolated segmentshave been followes. In every instance uptake is cumulative withthe greatest accumulation in the root tip. There are again largespecific differences but of a reverse order; uptake is leastfor P. sativum and greatest for S. vulgare. For these two speciesand Z. mays, it is concluded that the magnitude of the equi-effectiveconcentration required to halve root elognation is dependenton the level of accumulation rather than on the reaction atcell level: the cells of H. annuus are more sensitive.     

Photomorphogenic Regulation of Chloroplast Replication in Euglena: ENHANCED LOSS OF CHLOROPLAST DNA IN RED LIGHT

Chloroplast replication in Euglena gracilis is specifically inhibited by ultraviolet light and the effect is photoreactivable.     

New Methods for Characterization of Complex Receptor Systems Involving 3 or More Binding Sites: Application to Brain Opiate Receptors

Abstract

The problems of evaluating results based on the analysis of complex binding models are considered and new methods are proposed to provide independent confirmation of the existence of multiple sites. A new plotting format for the results from experiments involving two ligands is introduced, and its utility is demonstrated for a) finding initial estimates for nonlinear least squares curve fitting; b) presenting the results of multiple experiments; and c) giving a new means for evaluating the significance of a third site. The general problem of finding initial estimates for models involving three classes of sites, and strategies for using nonlinear least squares curve fitting algorithms to optimize the fit are considered.     

Xanthine-7-Ribosides as Adenosine Al Receptor Antagonists: Further Evidence for Adenosine's Anti Mode of Binding

Abstract

The synthesis and A₁ adenosine receptor affinity of some xanthine-7-ribosides is described. It appears that these compounds are A, receptor antagonists. The orientation of the ribose moiety, as determined by ′H-NMR spectroscopy and theoretical chemical calculations, is compared with the orientation of the ribose in the agonist adenosine. Implications for the syn vs anti modes of binding to the receptor are discussed.     

Evidence for Glucocorticoid Target Cells in the Rat Optic Nerve. Physicochemical Characterization of Cytosol Binding Sites

Abstract: Cytosolic dexamethasone (DEX) binding sites were studied in the Wallerian-degenerating rat optic nerve (ON), a tissue that is rich in neuroglial cells but devoid of neuronal perikarya and processes. For comparison, hippocampal (HI) and anterior pituitary (AP) cytosols were studied in parallel. Binding sites in these three tissues were found to be quite similar in almost all respects. The sites have a high affinity for DEX ( K _D= 2.5–3.5 n M ), are present at a high concentration ( B _max= 360–365 fmol/mg cytosol protein), and possess a binding specificity typical of glucocorticoid receptors in other organs. Most experiments supported the assumption of a single DEX-binding species in each tissue. Saturation analyses consistently yielded linear Scatchard plots over the range of DEX concentrations tested. Density gradient centrifugation in each case revealed a single peak with a sedimentation coefficient of 7–8S at low ionic strength and 4–4.5S in the presence of 0.3 M KCl. Isoelectric focusing similarly localized most of the binding in each cytosol to a single large peak with an isoelectric point of approximately 6.0. Dissociation rate determinations, on the other hand, suggested the possibility of two different binding sites in each tissue. These studies show that glucocorticoid binders present in cells of the ON possess the same characteristics as the cytoplasmic receptors found in HI, AP, and other recognized glucocorticoid target tissues.     

Distribution of Unlinked Receptor Sites for Transposed Ac Elements from the Bz-M2(ac) Allele in Maize

We have shown before that the Ac element from the maize bz-m2(Ac) allele, located in the short arm of chromosome 9 (9S), transposes preferentially to sites that are linked to the bz donor locus. Yet, about half of the Ac transpositions recovered from bz-m2(Ac) are in receptor sites not linked to the donor locus. In this study, we have analyzed the distribution of those unlinked receptor sites. Thirty-seven transposed Ac (trAc) elements that recombined independently of the bz locus were mapped using a set of wx reciprocal translocations. We found that the distribution of unlinked receptor sites for trAs was not random. Ten trAcs mapped to 9L, i.e., Ac had transposed to sites physically, if not genetically, linked to the donor site. Among chromosomes other than 9, the Ac element of bz-m2(Ac) appeared to have transposed preferentially to certain chromosomes, such as 5 and 7, but infrequently to others, such as 1, the longest chromosome in the maize genome. The seven trAc elements in chromosome 5 were mapped relative to markers in 5S and 5L and localized to both arms of 5. We also investigated the transposition of Ac to the homolog of the donor chromosome. We found that Ac rarely transposes from bz-m2(Ac) to the homologous chromosome 9. The clustering of Ac receptor sites around the donor locus has been taken to mean that a physical association between the donor site and nearby receptor sites occurs during transposition. The preferential occurrence of 9L among chromosomes harboring unlinked receptor sites would be expected according to this model, since sites in 9L would tend to be physically closer to 9S than sites in other chromosomes. The nonrandom pattern seen among the remaining chromosomes could reflect an underlying nuclear architecture, i.e., an ordering of the chromosomes in the interphase nucleus, as suggested from previous cytological observations.     

The Interaction of Na+ and K+ in Voltage-gated Potassium Channels : Evidence for Cation Binding Sites of Different Affinity

Voltage-gated potassium (K⁺) channels are multi-ion pores. Recent studies suggest that, similar to calcium channels, competition between ionic species for intrapore binding sites may contribute to ionic selectivity in at least some K⁺ channels. Molecular studies suggest that a putative constricted region of the pore, which is presumably the site of selectivity, may be as short as one ionic diameter in length. Taken together, these results suggest that selectivity may occur at just a single binding site in the pore. We are studying a chimeric K⁺ channel that is highly selective for K⁺ over Na⁺ in physiological solutions, but conducts Na⁺ in the absence of K⁺. Na⁺ and K⁺ currents both display slow (C-type) inactivation, but had markedly different inactivation and deactivation kinetics; Na⁺ currents inactivated more rapidly and deactivated more slowly than K⁺ currents. Currents carried by 160 mM Na⁺ were inhibited by external K⁺ with an apparent IC₅₀ <30 μM. K⁺ also altered both inactivation and deactivation kinetics of Na⁺ currents at these low concentrations. In the complementary experiment, currents carried by 3 mM K⁺ were inhibited by external Na⁺, with an apparent IC₅₀ of ∼100 mM. In contrast to the effects of low [K⁺] on Na⁺ current kinetics, Na⁺ did not affect K⁺ current kinetics, even at concentrations that inhibited K⁺ currents by 40–50%. These data suggest that Na⁺ block of K⁺ currents did not involve displacement of K⁺ from the high affinity site involved in gating kinetics. We present a model that describes the permeation pathway as a single high affinity, cation-selective binding site, flanked by low affinity, nonselective sites. This model quantitatively predicts the anomalous mole fraction behavior observed in two different K⁺ channels, differential K⁺ and Na⁺ conductance, and the concentration dependence of K⁺ block of Na⁺ currents and Na⁺ block of K⁺ currents. Based on our results, we hypothesize that the permeation pathway contains a single high affinity binding site, where selectivity and ionic modulation of gating occur.     

Studies in the Physiological Action of 2,2-Dichloropropionic Acid: II. THE EFFECTS OF LIGHT AND TEMPERATURE ON THE FACTORS RESPONSIBLE FOR THE INHIBITION OF GROWTH

When Lemna minor and Salvinia natans, grown in a constant environment,are subjected to sub-lethal concentrations of 2,2-dichioropropionicacid (DCPA), the relative growth-rates are progressively reduced.These cumulative reductions, which are greater for S. natans,are correlated with decreases in (1) the rate of leaf or frondformation, (2) the mean area per leaf or frond, and (3) thenet assimilation rate. Of these components, the first is themost important and the third is the least. The effects of light intensity (300, 600, 900 f.c.), temperature(20, 25, and 30°C), and concentration of DCPA on both therelative growth-rate and rate of leaf or frond multiplicationhave been examined in multi-factorial experiments. Over theconcentration range selected (100, 200, and 400 mg/l for S.natans and 100, 300, and 6oo mg/l for L. minor) there are positiveeffects of light intensity, temperature, and concentration.For each concentration the order of the depression is maximalunder a combination of the highest temperature and the greatestintensity. Using radioactive DCPA it has been established that uptake isalso a cumulative process, and that S. natans has a greatercapacity to absorb DCPA. The rate of uptake is independent ofthe light intensity but increases with temperature and concentration. DCPA brings about morphological and structural changes. In S.natans, many of the leaves become submerged and the proportionis positively dependent on light, temperature, and concentration.This failure to float is associated with a reduction in thedensity of epiderrnal hairs. It is concluded that the inhibitory effects of DCPA are maximalunder conditions which are optimal for both meristematic activityand accumulation.     

The Role of the Coleoptile Apex in Controlling Organ Elongation: II. EFFECTS OF AUXIN SUBSTITUTION AND AUXIN TRANSPORT INHIBITORS ON DECAPITATED COLEOPTILES

The most widely quoted evidence that auxin controls coleoptileelongation is that auxin applied to the cut surface of a coleoptilecan obviate the effects of decapitation. A quantitative studyof this old observation shows that exogenously applied auxinonly overcomes the effects of decapitation if supplied at unphysiologicallyhigh concentrations. Furthermore, when a ring of an auxin transportinhibitor is applied just beneath the apex of an intact coleoptile,so that auxin supplied from the apex is abolished, the growthrate of the coleoptile is not reduced significantly. These experimentssuggest that coleoptile elongation is not dependent on auxintransported from the apex. Key words: Coleoptile, auxin, transport inhibitors     

Selective Protection of Benzomorphan Binding Sites Against Inactivation by N-Ethylmaleimide. Evidence for k-Opioid Receptors in Frog Brain

Selective binding of [3H]bremazocine and [3H]-ethylketocyclazocine to kappa-opioid receptor sites in frog (Rana esculenta) brain membranes is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide (NEM). Pretreatment of the membranes with kappa-selective compounds [ethylketocyclazocine (EKC), dynorphin (1-13), or U-50,488H] but not with [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO; mu specific ligand) or [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DADLE; delta specific ligand) strongly protects the binding of the radioligands against NEM inactivation. These results provide more evidence for the existence of kappa-opioid receptors in frog brain. The relatively high concentrations of NEM that are needed to decrease the specific binding of [3H]bremazocine together with the observation of an almost complete protection of its binding sites by NaCl suggest that bremazocine may act as an opioid antagonist in frog brain.