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 w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">7, chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 and the Drosophila melanogaster/chicken hybrid receptors SAD/w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 and ALS/w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2. No agonist action of NTX (0.1–100 w7lvf6jd/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) was observed on chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">7, chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 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 (w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">7) or heteromeric (w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2) nicotinic AChRs. Block by NTX of the chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">7, chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 and the SAD/w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 and ALS/w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">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.     

Red-light- and gibberellic-acid-enhanced α-galactosidase activity in germinating lettuce seeds,cv. Grand Rapids

Dry lettuce (Lactuca sativa L.) w38j0408843m48/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">seedsw38j0408843m48/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> (achenes) contain w38j0408843m48/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-galactosidase (EC 3.2.122) at a level which is maintained in the imbibed dormant state in darkness. Both red light (R) and gibberellic acid promote an increase in enzyme activity several hours prior to the completion of germination. Germination and enzyme activity are not essentially linked, however, for the latter can increase while the former is inhibited. w38j0408843m48/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-Galactosidase activity increases within the cotyledons and the endosperm following R stimulation, but the axis is essential to perceive the stimulus and to promote and maintain the increase in enzyme activity. A diffusible factor (or factors) is produced by and-or released from irradiated axes, and it migrates to the cotyledons (and possibly endosperm) to promote the increase in w38j0408843m48/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-galactosidase activity. Gibberellic acid, particularly in the presence of benzyladenine, can replace the requirement for irradiated axes.Abbreviations GA₃ gibberellic acid - R red light     

Qualitative Analysis of the Carbohydrate Composition of Apolipoprotein H

The specific binding of digoxigenin-labeled lectins to carbohydrate moieties is used to characterize the carbohydrate chains bound to apolipoprotein H. Our results show that apolipoprotein H is rich in sialic acid linked w6871747j567vm06/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">(2–6) to galactose or N-acetylgalactosamine. Sialic acid is not w6871747j567vm06/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">(2–3)-linked to galactose. Galactose is w6871747j567vm06/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">(1–4)-linked to N-acetylglucosamine and w6871747j567vm06/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">(1–3)-linked to N-acetylgalactosamine. High-mannose N-glycan chains are barely detectable. After N-glycosidase F treatment the molecular weight is substantially reduced. The main band is 32,500 daltons. Carbohydrate O-linked chains, which are mainly represented by sialic acid, are w6871747j567vm06/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">(2–6)-linked to galactose or N-acetylgalactosamine. Galactose is also organized in O-linked chains and it is w6871747j567vm06/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">(1–4)-linked to N-acetylglucosamine and w6871747j567vm06/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">(1–3)-linked to acetylgalactosamine. Biochemical analysis of carbohydrate structures reveals that no specific carbohydrate complex is bound to a single isoform.     

Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens,not achievable with untransformed protoplasts

Summary Alternative methods for shoot regeneration in protoplast derived cultures were developed in Nicotiana paniculata and Physalis minima. In both species protoplast derived callus is not regeneratable to shoots by conventional methods, e.g. hormone treatment. Leaf discs and stem segments of N. paniculata and P. minima were incubated with Agrobacterium tumefaciens w320up407k8315/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">shooterw320up407k8315/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> strains harbouring pGV 2215 or pGV 2298 or wildtype strain B6S3. After 36 h of co-incubation protoplasts were prepared. (Leaf disc and stem segment cloning). Co-cultivation experiments were also undertaken with protoplasts of both species. Transformed clones, characterized by their hormone independent growth and octopine production, could be isolated after about two months. Transformation frequencies of w320up407k8315/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">leaf disc and stem segment cloningw320up407k8315/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> and co-cultivation experiments varied from 5×10^–3 to 5×10^–5. After about one year of cultivation on hormone-free culture medium, shoots could be recovered from colonies of N. paniculata, transformed by the strain harbouring pGV 2298. In protoplast derived colonies of P. minima, shoot induction was obtained only after transformation by bacteria carrying pGV 2215. This demonstrates the importance of the particular w320up407k8315/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">shooterw320up407k8315/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> mutant, as well as the response of the host plant. Transformed shoots of P. minima produced octopine, whereas octopine production in transformed shoots and callus of N. paniculata was undetectable after one year of cultivation, though T-DNA was still present in the plant genome. Transformed shoots of N. paniculata and P. minima do not produce any roots. Shoots of N. paniculata have an especially tumerous phenotype. Shoots of both species were successfully grafted to normal donor plants of N. tabacum.Abbreviations B5-h Gamborg medium without hormones (Gamborg 1968) - V47 protoplast medium (Binding 1974) - D2a protoplast medium (Li et al. 1980) - MS-h Murashige and Skoog medium without hormones (Murashige and Skoog 1962) Dedicated to Professor Dr. G. Melchers in occasion of his 80th birthday     

Localization of α-amylase isozymes within the endomembrane system of barley aleurone

Summary The localization of w75425707323/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts was studied using electron microscope immunocytochemistry. Antibodies were raised against total barley w75425707323/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase, i.e., w75425707323/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase containing both highisoelectric point (high-pI) and low-pI isoforms, as well as against purified high- and low-pI isoforms. All antibodies localized w75425707323/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase to the endoplasmic reticulum (ER) and Golgi apparatus (GApp) of the aleurone cell, and various controls showed that the labeling was specific for w75425707323/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase. Labeling of protein bodies and spherosomes, which are the most abundant organelles in this cell, was very low. There was no evidence that w75425707323/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase isoforms were differentially distributed within different compartments of the endomembrane system. Rather, both high- and low-pI isoforms showed the same pattern of distribution in ER and in the cis, medial, and transregions of the GApp. We conclude that in the Himalaya cultivar of barley, all isoforms of w75425707323/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase are transported to the plasma membrane via the GApp.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - GApp Golgi apparatus - PBS phosphate buffered saline - PCR partially coated reticulum - PM plasma membrane - TBS Tris buffered saline - TGN trans-Golgi network     

Centrin- and α-actinin-like immunoreactivity in the ciliary rootlets of insect sensilla

Summary Long ciliary rootlets are a characteristic feature of the dendritic inner segments of the sensory cells in insect sensilla. These rootlets are composed of highly ordered filaments and are regularly cross-striated. Collagenase digestion and immunohistochemistry reveal that the rootlets are probably not composed of collagen fibers. However, double-labeling experiments with phalloidin and anti-w5351/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-actinins show that antibodies to w5351/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-actinin react with the ciliary rootlets of the sensilla, but do not stain the scolopale, which is composed of actin filaments as visualized by phalloidin. Antibodies to centrin, a contractile protein isolated from flagellar rootlets of green algae, also stain the ciliary rootlets. Within the ciliary rootlets of insect sensilla, w5351/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-actinin may be associated with filaments other than actin filaments. The immunohistochemical localization of a centrin-like protein suggests that contractions probably occur within the rootlets. The centrin-like protein may play a role during the mechanical transduction or adaptation of the sensilla.     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Sources of nitrate in rivers draining sixteen watersheds in the northeastern U.S.: Isotopic constraints

The feasibility of using nitrogen and oxygenisotope ratios of nitrate (NO₃ ^–) forelucidating sources and transformations ofriverine nitrate was evaluated in a comparativestudy of 16 watersheds in the northeastern U.S.A. Stream water was sampled repeatedly at theoutlets of the watersheds between January andDecember 1999 for determining concentrations,w/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N values, and w/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸Ovalues of riverine nitrate.In conjunction with information about land useand nitrogen fluxes,w/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N_nitrate andw/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O_nitrate values providedmainly information about sources of riverinenitrate. In predominantly forested watersheds,riverine nitrate had mean concentrations ofless than 0.4 mg NO₃ ^–-N L^–1,w/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N_nitrate values of lessthan +5w/xxlarge8240.gif" alt="permil" align="MIDDLE" BORDER="0">, and w/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O_nitratevalues between +12 and +19w/xxlarge8240.gif" alt="permil" align="MIDDLE" BORDER="0">. This indicatesthat riverine nitrate was almost exclusivelyderived from soil nitrification processes withpotentially minor nitrate contributions fromatmospheric deposition in some catchments. Inwatersheds with significant agricultural andurban land use, concentrations of riverinenitrate were as high as 2.6 mg NO₃ ^–-NL^–1 with w/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N_nitratevalues between +5 and +8w/xxlarge8240.gif" alt="permil" align="MIDDLE" BORDER="0"> andw/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O_nitrate values generallybelow +15w/xxlarge8240.gif" alt="permil" align="MIDDLE" BORDER="0">. Correlations between nitrateconcentrations, w/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N_nitratevalues, and N fluxes suggest that nitrate inwaste water constituted a major, and nitrate inmanure a minor additional source of riverinenitrate. Atmospheric nitrate deposition ornitrate-containing fertilizers were not asignificant source of riverine nitrate inwatersheds with significant agricultural andurban land use. Although complementary studiesindicate that in-stream denitrification wassignificant in all rivers, the isotopiccomposition of riverine nitrate sampled at theoutlet of the 16 watersheds did not provideevidence for denitrification in the form ofelevated w/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁵N_nitrate andw/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">¹⁸O_nitrate values. Relativelylow isotopic enrichment factors for nitrogenand oxygen during in-stream denitrification andcontinuous admixture of nitrate from theabove-described sources are thought to beresponsible for this finding.     

Monensin inhibits the secretion of α-amylase but not polysaccharide slime from seedling tissues ofZea mays

Summary The effect of monensin on polysaccharide slime secretion by root tips of corn (Zea mays) was studied. Various treatment times and ionophore concentrations were tested: none resulted in inhibition of slime secretion. Because monensin changes the pH of the medium, its effect was also monitored in strongly buffered media and at different pH's. Even in such media, monensin did not inhibit slime secretion. We also measured the effect of the drug after a pulse with [³H]fucose or a pulse followed by a chase. The amount of labeled slimed secreted was not altered by the ionophore. However, 10w25ml7/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M monensin affected the development of root tips and drastically reduced their growth. We showed that monensin inhibits the secretion of w25ml7/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-amylase by the scutellum of the same plantlet. The importance of the nature of the secretory compound in relation to monensin inhibition of its secretion is discussed.Abbrevations Hepes N-2-hydroxyethylpiperazine-Nw25ml7/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-2-ethane-sul-fonic acid - Mes 2-(N-morpholino)ethane-sulfonic acid     

Tree-ring analysis and conifer growth responses to increased atmospheric CO2 levels

Summary Tree-ring data of naturally grown connifers were analyzed to evaluate the possibility of enhanced tree growth due to increased atmospheric CO₂. Tree cores were obtained from 34 sites in four different climatic regions in the northern hemisphere. In each of the four regions, the sampling sites were located along ecological gradients between the subalpine treeline and low elevations and, sometimes, the arid forest border. Growth trends after 1950, when the atmospheric CO₂ concentration increased by more than 30 w648r2364h106347/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">l·l^-1 indicate an increase in ring-widths at eight of the 34 sites. These chronologies were from sites which moderate temperature or water stress. In four cases the growth increase in the post-1950 period coincided with favorable climatic conditions. In the remaining four cases, the growth increase exceeded the upper bound response expected from CO₂ enrichment experiments with seedling conifer species. Therefore, increased growth in any of the tree-ring chronologies examined could not be solely attributed to higher atmospheric CO₂ concentrations.Major financial supporters: Swiss National Science Foundation (application no. 1.869-0.83); Swiss Federal Institute of Forestry Research, 8903 Birmensdorf, Switzerland; other financial supporters: Carbon Dioxide Research Division, U.S. Department of Energy under subcontract no. 11X-57507V with Martin Marietta Energy Systems, IncOperated by Martin Marietta Energy Systems, Inc., under contract DE-AC05-840R21400 with U.S. Department of Energy