Nicotinic receptor alpha7-subunits are coupled to the stimulation of nitric oxide synthase in rat dorsal root ganglion neurons

In dorsal root ganglia (DRG) intraganglionic communication takes place both among neurons and between neurons and satellite cells. One diffusible substance involved in this signalling is nitric oxide (NO), and acetylcholine (ACh) is a candidate for the stimulation of intraganglionic NO synthesis. DRG neurons react to ACh-receptor stimulation with NO-dependent cGMP production. Here, we investigated the role of the 7-subunit containing Ca²⁺-permeable nicotinic ACh receptors (nAChR) in this process. The 7-nAChR mRNA and the protein were expressed in virtually all lumbar DRG neurons as evidenced by laser-assisted cell picking and oligo cell RT-PCR, in situ hybridisation and immunohistochemistry. Strong 7-nAChR immunoreactivity was present in vanilloid receptor 1-immunoreactive, i.e. nociceptive, neurons. A neuronal production of NO in response to nicotine could be demonstrated in DRG slice preparations utilising the NO-sensitive fluorescent indicator diaminofluorescein diacetate (DAF-2DA). This stimulation of NO production was sensitive to inhibition of 7-nAChR by mecamylamine and -bungarotoxin, to inhibition of nitric oxide synthase (NOS) with L-NAME and L-NMMA, and to the blockade of voltage-operated Ca²⁺ channels by verapamil. The results show the presence of the 7-nAChR subunit in nociceptive rat DRG neurons and provide evidence for its coupling to NOS activation, indicating a role of this pathway in the intraganglionic communication in sensory ganglia.     

Local Perfusion of Nicotine Differentially Modulates Somatodendritic Dopamine Release in the Rat Ventral Tegmental Area After Nicotine Preexposure

We examined the effects of nicotine perfusion into the ventral tegmental area (VTA) on extracellular dopamine (DA) levels in rats using in vivo microdialysis. Local perfusion with nicotine for 80 min (10–100 M) modestly increased (105–131% of basal) the extracellular DA levels in the VTA of rats that had been pretreated with saline for 5 days. In animals that had been pretreated with nicotine for 5 days (0.3 mg/kg, s.c.), perfusion with nicotine for 80 min (10–100 M) dose-dependently increased the extracellular DA levels in the VTA of rats and did so to a greater extent than in saline-pretreated animals (125–171% of basal). Co-perfusion through the dialysis probe with 100 M mecamylamine, a nonselective nicotinic acetylcholine receptor (nAChR) antagonist, or 100 M dihydro--erythroidine, a high affinity and competitive nAChR antagonist, attenuated the enhancement of extracellular DA levels produced by 100 M nicotine alone. These results suggest that local nicotine challenge potentiated the somatodendritic DA release after nicotine preexposure by stimulation of high-affinity nAChRs in the VTA.     

Mapping of Exposed Surfaces of the Nicotinic Acetylcholine Receptor by Identification of Iodinated Tyrosine Residues

Here we report on the use of iodination of the membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue in order to define surface-exposed portions of the receptor molecule. Membrane-bound nAChR was ¹²⁵I-iodinated using the oxidation agent Iodo-Gen. The iodinated subunits were separated by preparative gel electrophoresis, desalted, and cleaved with trypsin. The resulting peptides were separated by reverse-phase HPLC and the radioactive peptides were identified by mass spectrometry and protein sequencing. For the -subunit, we identified five iodinated peptides containing the tyrosine residues Tyr17, Tyr74, Tyr365, Tyr372, and Tyr428. The surface exposition of these amino acids is in agreement with the four-transmembrane-segment model (4TM model) of the nAChR, but the assignment to the intra- or extracellular surface is doubtful. According to this model, the N-terminal portion of the receptor subunits including the iodinated residues Tyr17 and Tyr74 is extracellular and Tyr372 as a site of tyrosine phosphorylation is located on the cytoplasmic side. But since this latter residue is among the first to be iodinated using an immobilized iodination agent, its true position with respect to the membrane bilayer is not clear.     

Nicotine-related brain disorders: The neurobiological basis of nicotine dependence

Summary 1. This paper was written at a moment when the dependence liability of nicotine, the psychoactive component from tobacco, was the center of a dispute between the tobacco manufacturing companies and the scientific community (Nowak, 1994a–c). Without being comprehensive, it tries to summarize evidence compiled from several disciplines within neuroscience demonstrating that nicotine produces a true psychiatric disease, behaviorally expressed as dependence to the drug (American Psychiatric Association, 1994). Nicotine dependence has a biological substratum defined as neuroadaptation to nicotine.2. The first part of the article defines terms such as abuse, tolerance, dependence, and withdrawal. It discusses clinical and experimental facts at the whole-organism level, showing that animals and humans will seek and self-administer nicotine because of its rewarding properties.3. The second part discusses the neurobiological basis of neuroadaptation to nicotine. It presents information on neuroanatomical circuits which may be involved in nicotine-related brain disorders, such as the mesocorticolimbic pathway and the basal forebrain-frontal cortex pathway. It also discusses work from several laboratories, including our own, that support the notion of a molecular basis for neuroadaptative changes induced by nicotine in the brain of a chronic smoker.4. Although still under experimental scrutiny, the hallmark of neuroadaptation to nicotine is up-regulation of nicotinic receptors, possibly due to nicotine-induced desensitization of their function (Markset al., 1983; Schwartz and Kellar, 1985). A correlation between these plastic changes and the behavioral data obtained from animal and human experiments is still needed to understand dependence to nicotine fully.     

Functional Neurochemical Evidence for the Presence of Presynaptic Nicotinic Acetylcholine Receptors at the Terminal Region of Myenteric Motoneurons: A Study with Epibatidine

The aim of this study was to verify the presence of presynaptic nicotinic acetylcholine receptors (nAChRs) at the terminals of myenteric motoneurons using a potent and highly selective nicotinic agonist, epibatidine. We examined contraction, and release of [³H]ACh on a guinea-pig longitudinal muscle strip preparation. First, we compared the ability of epibatidine and nicotine to induce isometric contraction and found epibatidine (EC₅₀ = 23.1 nM) to be 300-fold more potent than nicotine (EC₅₀ = 7.09 M). The release and contraction induced by 30 nM epibatidine were inhibited by the nicotinic antagonist mecamylamine (3 M) and the Na1-channel blocker TTX (1 M), indicating that the effects are mediated via nAChRs and are fully dependent on the propagation of action potentials. Atropine (0.1 M) significantly increased the [³H]ACh release but could not block contraction suggesting that a substantial part of the response develops via a noncholinergic mechanism. Epibatidine at a higher concentration (300 nM) induced contraction, which was only partly (45%) inhibited by TTX (1 M). The TTX-resistant contraction, however, was completely blocked by mecamylamine (3 M). Our data provide functional neurochemical evidence for the existence of presynaptic nAChRs at myenteric motoneuron terminals and suggest that these receptors can be activated only/by a higher concentration of agonists.     

The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

Carnivora: the primary structure of the major hemoglobin component from adult European lynx (Lynx lynx, Felidae).

The complete primary structure of the major hemoglobin component from the adult European lynx (Lynx lynx) is presented. Presence of two hemoglobin components and three chains, A, B, and , identified by gel electrophoresis. The purification of the globin chains achieved by ion-exchange chromatography. The globin chains were digested with trypsin. The peptide generated were purified by reversed-phase HPLC. Sequencing of the native chains up to 42 cycles and of the tryptic peptides were deduced by Edman degradation in liquid- and gasphase sequencer. The primary structure established aligned with those of human Hb-A. The comparison of lynx globin chains with other representatives of the Felidae, lion, tiger, jaguar, leopard, and cat revealed high homology.Deceased on May 27, 1989.     

Liquid chromatography “on-flow” 1H nuclear magnetic resonance on native glycosphingolipid mixtures together with gas chromatography/mass spectrometry on the released oligosaccharides for screening and characterisation of carbohydrate-based antigens from pig lungs

Glycosphingolipids were prepared from pig lung and pooled into two fractions with (i) 3 sugar residues, and (ii) 3 sugar residues. Oligosaccharides were prepared and used for gas chromatography, gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The glycolipid fractions i and ii were further characterised and purified using a novel method based on high performance liquid chromatography on-flow proton nuclear magnetic resonance. The LC on-flow NMR technique showed good chromatographic separation and gave NMR spectral information which could be used as guidance for pooling of the separated mixture glycolipids. Conventional ¹H NMR, thin layer immunostaining, gas chromatography, gas chromatography/mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry were used to characterise the glycolipids and to validate LC-NMR spectral data.     

High crossability of wild barley (Hordeum spontaneum C. Koch) with bread wheat and the differential elimination of barley chromosomes in the hybrids

Four bread wheat (Triticum aestivum L.) cultivars, Aobakomugi, Chinese Spring, Norin 61 and Shinchunaga, were pollinated with five barley lines/cultivars consisting of three cultivated barley (Hordeum vulgare L.) lines, Betzes, Kinai 5 and OHL089, and two wild barley (Hordeum spontaneum C. Koch) lines, OUH602 and OUH324. Crossability, expressed as the percentage of embryo formation, varied from 0 to 55.4% among the cross combinations. The two wild barley lines generally had a higher crossability than the previously reported best pollinator, Betzes, and some Japanese wheat cultivars were better as the female parent than Chinese Spring. Ninety four hybrid plants were obtained from 250 embryos cultured, and their somatic chromosome numbers ranged from 21 to 36. Eighteen plants were mosaic in chromosome number. Twenty one-chromosome plants appeared most frequently (45.7%) followed by 28-chromosome plants (14.9%). C-banding analysis revealed that elimination of barley chromosomes was mainly responsible for the occurrence of aneuploid plants. In hypoploids derived from Betzes-crosses, chromosome 5 was preferentially eliminated as previously reported, while in hypoploids derived from OUH602-crosses, chromosome 4 was preferentially eliminated. The wild barley line OUH602 may be a useful parent for producing a new wheat-barley addition set because of its high crossability with wheat and a different pattern of chromosome elimination.     

Differential desensitization properties of rat neuronal nicotinic acetylcholine receptor subunit combinations expressed inXenopus laevis oocytes

Summary 1. Chronic administration of nicotine up-regulates mammalian neuronal nicotinic acetylcholine receptors (nAChRs). A key hypothesis that explains up-regulation assumes that nicotine induces desensitization of receptor function. This is correlated with behaviorally expressed tolerance to the drug.2. The present experiments were conducted to: (a) obtain information on the nicotine-induced desensitization of neuronal nAChR function, a less understood phenomenon as compared to that of the muscle and electric fish receptor counterparts; (b) test the hypothesis that different receptor subunit combinations exhibit distinct desensitization patterns.3.Xenopus laevis oocytes were injected with mRNAs encoding rat receptor subunits2,3, or4 in pairwise combination with the2 subunit. The responses to various concentrations of acetylcholine (ACh) or nicotine were analyzed by the two electrode voltage clamp technique.4. Concentration-effect curves showed that nicotine was more potent than ACh for all the receptor subunit combinations tested. Only the42 combination exhibited a depression of the maximum effect at concentrations higher than 20µM nicotine.5. After a single nicotine pulse, receptor desensitization (calculated as a single exponential decay) was significantly slower for42 than for either32 or22.6. Concentrations of nicotine that attained a near maximum effect were applied, washed, and re-applied in four minute cycles. The responses were calculated as percentages of the current evoked by the initial application. Following 16 minutes of this protocol, the42 combination showed a greater reduction of the original response as compared to the22 and32 subunit combinations. Taking points 5 and 6 together, these experiments suggest that the42 receptor subtype desensitizes at a slower rate and remains longer in the desensitized state.7. Because42 is the main receptor subunit combination within the brain and is up-regulated by nicotine, our data may be important for understanding the molecular basis of tolerance to this drug.     

Osmotic Forces and Gap Junctions in Spreading Depression: A Computational Model

In a computational model of spreading depression (SD), ionic movement through a neuronal syncytium of cells connected by gap junctions is described electrodiffusively. Simulations predict that SD will not occur unless cells are allowed to expand in response to osmotic pressure gradients and K⁺ is allowed to move through gap junctions. SD waves of [K⁺]_out 25 to 60 mM moving at 2 to 18 mm/min are predicted over the range of parametric values reported in gray matter, with extracellular space decreasing up to 50%. Predicted waveform shape is qualitatively similar to laboratory reports. The delayed-rectifier, NMDA, BK, and Na⁺ currents are predicted to facilitate SD, while SK and A-type K⁺ currents and glial activity impede SD. These predictions are consonant with recent findings that gap junction poisons block SD and support the theories that cytosolic diffusion via gap junctions and osmotic forces are important mechanisms underlying SD.     

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken 7, chicken 42 and the Drosophila melanogaster/chicken hybrid receptors SAD/2 and ALS/2. No agonist action of NTX (0.1–100 M) was observed on chicken 7, chicken 42 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (7) or heteromeric (42) nicotinic AChRs. Block by NTX of the chicken 7, chicken 42 and the SAD/2 and ALS/2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

Automated NMR determination of protein backbone dihedral angles from cross-correlated spin relaxation

The simultaneous interpretation of a suite of dipole-dipole and dipole-CSA cross-correlation rates involving the backbone nuclei ¹³C, ¹H,¹³CO, ¹⁵N and ¹H^N can be used to resolve the ambiguities associated with each individual cross-correlation rate. The method is based on the transformation of experimental cross-correlation rates via calculated values based on standard peptide plane geometry and solid-state ¹³CO CSA parameters into a dihedral angle probability surface. Triple resonance NMR experiments with improved sensitivity have been devised for the quantification of relaxation interference between ¹H(i)-¹³C(i)/¹⁵N(i)-¹H^N(i) and ¹H(i–1)-¹³C(i–1)/¹⁵N(i)-¹H^N(i) dipole-dipole mechanisms in ¹⁵N,¹³C-labeled proteins. The approach is illustrated with an application to ¹³C,¹⁵N-labeled ubiquitin.     

Effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats

Endotoxin shock is characterized by systemic hypotension, hyporeactiveness to vasoconstrictors and acute lung edema. A nitric oxide synthase (NOS) inhibitor, N^G-monomethyl-L-arginine (L-NMMA) has been shown to be effective in reversing acute lung injury. In the present study, we evaluated the effects of NOS blockade by different mechanisms on the endotoxin-induced changes. In anesthetized rats, lipopolysaccharide (LPS,Klebsiella pneumoniae) was administered intravenously in a dose of 10 mg/kg. LPS caused sustained systemic hypotension accompanied by an eightfold increase of exhaled NO during an observation period of 4 h. After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expressions of inducible NOS (iNOS), interleukin-1 (IL-1) and tumor necrosis factor--(TNF-). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1 and TNF- were absent. Four hours after LPS, the mRNA expressions of iNOS and IL-1 were still significantly enhanced, but TNF- was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial damage and interstitial edema. Various NOS inhibitors were given 1 h after LPS administration. These agents included N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), a constitutive NOS and iNOS inhibitor; S,S-1,4-phenylene-bis-(1,2-ethanedinyl) bis-isothiourea dihydrobromide (1,4-PBIT, 10 mg/kg), a relatively specific iNOS inhibitor, and dexamethasone (3 mg/kg), an inhibitor of iNOS expression. These NOS inhibitors all effectively reversed the systemic hypotension, reduced the exhaled NO concentration and prevented acute lung injury. The LPS-induced mRNA expressions of iNOS and IL-1 were also significantly depressed by these NOS inhibitors. Our results suggest that NO production through the iNOS pathway is responsible for endotoxin-induced lung injury. Certain cytokines such as IL-1 are possibly involved. These changes are minimized by NOS inhibitors through different mechanisms.     

A novel glycosphingolipid expressed in pig kidney: Galα1-3Lewisx hexaglycosylceramide

Immunodetection of thin layer chromatograms of neutral glycosphingolipids of pig kidney cortex with a polyclonal antibody directed against the Gal1-3Gal determinant revealed several glycosphingolipids reacting with different intensities. A minor glycosphingolipid was isolated by preparative high performance thin layer chromatography. It was characterized as a type 2 hexaglycosylceramide with the following structure Gal1-3Gal1-4(Fuc1-3)GlcNAc1-3Gal1-4Glc1-Cer by fast atom bombardment- and desorption-chemical ionization-mass spectrometry, methylation analysis and hydrolysis with -galactosidase followed by immunostaining with an anti-Lewisx monoclonal antibody. The proton NMR spectrum was found compatible with the proposed structure. Two other glycosphingolipids carrying the new determinant were partially characterized as an octa- and a branched-dodecaglycosylceramide. The expression of the Gal1-3Lewisx determinant appeared to be developmentally regulated as it increased with age. The characterization of Gal1-3Lex in pig kidney indicates a new epitope capable of recognition by human natural antibodies in the context of xenotransplantation of pig organs to man. It also adds new members to the family of Lex-based glycolipids. Abbreviations: HPTLC, high performance thin layer chromatography; FAB-MS, fast atom bombardment mass spectrometry; DCI, desorption-chemical ionization; Me2SO-d6, hexadeuterated dimethyl sulfoxide     

Structural analyses of new tri- and tetrasaccharides produced from disaccharides by transglycosylation of purified Trichoderma viride β-glucosidase

A new -glucosidase was partially purified from Trichoderma viride cellulase. This -glucosidase catalyzed a transglycosylation reaction of cellobiose to give -D-Glc-(16)--D-Glc-(14)-D-Glc (1, yield: 18.8%) and -D-Glc-(16)--D-Glc-(16)--D-Glc-(14)-D-Glc (2, 3.7%), regioselectively. Furthermore, the enzyme regioselectively converted laminaribiose and gentiobiose into -D-Glc-(16)--D-Glc-(13)-D-Glc (3, 15.3%) and -D-Glc-(16)--D-Glc-(16)-D-Glc (4, 20.2%), respectively. The structures (1–4) of the products were determined by ¹H and ¹³C NMR spectroscopies. This high regio- and stereoselectively of the -glucosidase could be applied for oligosaccharide synthesis.     

The pyridine-nucleotide cycle in tobacco

In order to elucidate the NAD-recycling pathway the following enzyme activities have been characterized in different tobacco tissues and in tomato root: NAD pyrophosphatase, nicotinamide mononucleotide (NMN)/nicotinic acid mononucleotide (NaMN) glycohydrolases, nicotinamidase and nicotinic acid phosphoribosyltransferase. The investigations were performed with protein extracts purified by gel filtration and enzymatic activities were determined by high-performance liquid chromatography methods. The kinetic parameters of the different enzymes from tobacco root and their specificity are reported. The data are in favor of the so-called pyridine-nucleotide cycle VI (NADNMNnicotinamidenicotinic acidNaMNnicotinic acid adenine dinucleotideNAD). In the nicotine-producing tobacco root a further direct route leading from NaMN to nicotinic acid is proposed. These data are reconciled with the assumption that it is nicotinic acid which is provided by the pyridine-nucleotide cycle for the synthesis of nicotine.Abbreviations HPLC high-performance liquid chromatography - Na nicotinic acid - NaAD nicotinic acid adenine dinucleotide - NaMN nicotinic acid mononucleotide - NMN nicotinamide mononucleotide - PRPP 5-phosphoribosyl-1-pyrophosphate This contribution is dedicated to Professor Augustin Betz on the occasion of his 65^th birthday     

Molecular evolution of the nicotinic acetylcholine receptor: An example of multigene family in excitable cells

An extensive phylogenetic analysis of the nicotinic-acetylcholine-receptor subunit gene family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTAL V and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I-the neuronal -bungarotoxin binding-site subunits (7, 8); III-the neuronal nicotinic subunits (2–6, 2–4), which also contain the muscle acetylcholine-binding subunit (1); and IV—the muscle non- subunits (1, , ). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.Abbreviations -Bgt -bungarotoxin - ACh acetylcholine - MP maximum of parsimony - MYA million years ago - NJ neighbor-joining - nAChR nicotinic acetylcholine receptor Correspondence to: N. Le Novère     

A 42 bp fragment of the pmas1′ promoter containing an ocs-like element confers a developmental,wound- and chemically inducible expression pattern

Synthesis of mannopine in plant tissues infected with Agrobacterium tumefaciens is controlled by a divergent promoter (pmas2 and pmas1) that in 479 bp contains all the cis-acting elements necessary to direct tissue-specific and wound-inducible expression. In this report, using transgenic tobacco plants harboring a pmas1--glucuronidase (GUS) gene fusion, we investigated the developmental expression pattern directed by pmas1 in the early stages of development and the responses of pmas1 to different chemical inducers. It was found that this promoter can respond to auxins, cytokinins, methyl jasmonate (MJ), salicylic acid (SA) and its analogue 2,6-dichloroisonicotinic acid (iNA). Treatment with chemical inducers also showed that the effects of iNA are organ-dependent, that wound-induction is a complex response mediated by at least two different chemical signals, and that MJ stimulates changes in the tissue-specific and developmental expression pattern directed by the pmas1 promoter. Using chimeric promoters we demonstrate that an ocs-like element (ocs+1) directs MJ responses in an orientation-dependent manner and that sequences around the ocs+1 are important to maintain the inducible and developmental properties of this cis-regulatory element.     

The glycolipid specificity ofErythrina cristagalli agglutinin

The interaction of¹²⁵I-labeledErythrina cristagalli agglutinin (ECA) with neutral glycosphingolipids on thin layer chromatograms was examined by the overlay technique followed by radioautography. The lectin bound topara-globoside with a sensitivity about 10 times higher than to lactosylceramide or globoside, in agreement with the specificity of the lectin forN-acetyllactosamine. The lower limit of detection ofpara-globoside was about 0.66 nmol. The specific binding of ECA to this glycolipid was confirmed by a highly sensitive enzyme-linked lectin assay (ELLA), utilizing the horseradish peroxidase-avidin-biotin system for detection of bound lectin. Overlays of neutral glycosphingolipid extracts from human erythrocyte membranes and from human granulocytes with ECA demonstrated that the lectin can be employed for the detection of small amounts ofpara-globoside in biological materials also in the presence of excess globoside. No staining was obtained when thin layer chromatograms of neutral glycosphingolipid extracts from rabbit erythrocyte membranes were overlayed with¹²⁵I-ECA. Afterin situ treatment of the chromatograms with -galactosidase, the lectin bound to several components, one of which had a mobility corresponding to that of the pentahexosylceramide Gal3Gal4GlcNAc3Gal4Glc1Cer, the major neutral glycosphingolipid of rabbit erythrocytes, thus providing further evidence for the specificity of ECA forpara-globoside.Abbreviations GSL glycosphingolipid(s) - CDH lactosylceramide, Gal4Glc1Cer - CTH trihexosylceramide, Gal4Gal4Glc1Cer - GLOB globoside, GalNac3Gal4Gal4Glc1Cer - PG para-globoside, Gal4GlcNAc3Gal4Glc1Cer - AsG_M1 asialo-G_M1, Gal3GalNAc4Gal4Glc1Cer - FORS Forsmann antigen, GalNAc3GalNAc3Gal4Gal4Glc1Cer - CPH pentahexosylceramide, Gal3Gal4GlcNAc3Gal4Glc1Cer - ECA Erythrina cristagalli agglutinin - SBA soybean agglutinin - PBS phosphate-buffered saline - PVP-40 polyvinylpyrrolidone M.W. 40000 - BSA bovine serum albumin - HRP-avidin horseradish peroxidase conjugated to avidin - ELLA enzyme-linked lectin assay - ELISA enzyme-linked immunosorbent assay - PMNL polymorphonuclear leukocytes - HPTLC high performance thin layer chromatography