Photoreactive dTTP Analogues as Substrates for Thermostable DNA Polymerase from Thermus thermophilus B35

Substrate properties of several dTTP analogues bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group attached at position 5 of uracil through linkers of various lengths, dTTP–NAB-x-dUTP (where x = 2, 4, 7–13 is the number of atoms in the linker), were studied. All the analogues are substrates for thermostable Thermus thermophilus B35 DNA polymerase in the elongation reaction of the 5-³²P-labeled primer–template complex. The kinetic parameters of some of the analogues were determined and compared with those of natural dTTP. It was shown that an increase in the linker length results in a higher efficiency of the analogue. The incorporation of NAB-x-dUP residues into the 3 primer end did not impede further elongation of the chain in the presence of natural dNTP.     

Interactions of growth inhibitory factor with hydroxyl and superoxide radicals

To show the effects of growth inhibitory factor (Cu₄Zn₃MT-III) involved in the scavenging of reactive oxygen species (ROS), a pulse radiolytic study was employed using N₂O-saturated Cu₄Zn₃MT-III aqueous solutions. It was demonstrated that the oxidizing OH radical efficiently reacted with Cu₄Zn₃MT-III by forming a thiyl radical RS with a second-order constant of 1.46×10¹¹ mol l^–1s^–1, which was determined by competition kinetics against KSCN. The thiyl radical RS reacted rapidly and reversibly with a thiolate in Cu₄Zn₃MT-III to form radical anion RSSR ^– with a constant of 1.65×10⁹ mol lL^–1s^–1 per thiolate, while the constant of the decay of this radical anion was 2.72×10⁵ s^–1, and the equilibrium constant of the formation for RSSR ^– was 6.08×10³ mol^–1 l. These values were close to those of Cd₅Zn₂MT-II. The SOD activity of Cu₄Zn₃MT-III to quench O₂ ^–was assayed by the riboflavine-methionine-nitrobluetetrazolium (NBT) method which catalyzed the dismutation of superoxide (O₂ ^–) at pH 7.8 with an IC₅₀ value of 1.50×10^–6 M for Cu₄Zn₃MT-III and 1.62×10^–6 M for Cd₅Zn₂MT-II. Additionally, the down-regulation of GIF may be a main factor in the decrease of the scavenging ability for the free OH and O₂ ^–radicals, which is possibly associated with the pathogenesis of neurodegenerative disease.     

Water flux through a semi-deciduous forest grove of the Orinoco savannas

Water relations were analysed in a semi-deciduous forest grove occurring in the oxisols of the Orinoco savannas. This grove has a shallow unconsolidated ironstone cuirass, which is overlaid by a sandy loam layer (0.0–0.5 m) that contains more than 90% of the grove forest root phytomass. Evapotranspiration and through drainage were calculated by using data from the soil profile as related to physical characteristics of the site root zone, hydraulic conductivity, volumetric water content and potential hydraulic gradient. Mean annual evapotranspiration was 783 mm year^–1 and annual through drainage below the root zone was 14% (162 mm year^–1) of the gross rainfall. This drainage recharged the 42% of the annual saturation deficit of the water table. Similar mean annual evapotranspiration (770 mm year^–1) was also calculated by using the water balance components. The mean daily coupling omega factor () between the grove canopy and the surrounding atmosphere indicated that a high degree of coupling (=0.14±0.16) occurs in the grove and evapotranspiration was mainly controlled by surface conductance. As the dry season proceeded, the soil saturation deficit () increased rapidly resulting in a threshold surface conductance (0.030–0.005 m s^–1) for ranging from 0.05 to 0.10. Hypotheses to explain the omnipresence of perennial species in the wide range of physical conditions in neotropical savannas are discussed.     

A novel eye morphology induced by a P element in somatic tissue of Drosophila melanogaster.

Summary We found a specific eye morphology designated as Square, which is induced when some Drosophila melanogaster strains harboring P elements are crossed with the 2–3 strain carrying a modified P element, P[ry ⁺, 2–3], which produces transposase in somatic tissue. This phenotype was dominant and also induced in the reciprocal crosses. Square was induced when the 2–3 strain was crossed with Q and M strains such as the sn^w (M) strain carrying three small P elements but not with P strains. Inheritance of Square was also tested and its phenotype was not transmitted to the next generation. These results suggest that Square is caused by the transposition of P elements in somatic cells.     

New ganglioside analogs that inhibit influenze virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac)2-S-6)Glc-(1-1)Ceramide (1) and the GM3 analog Neu5Ac(2-S-6)Gal-(1–4)Glc(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent K_i value of 2.8×10^–6 and 1.5×10^–5 M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac-(2-S-6)Gal-(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (K_i =1.0×10^–4 M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were nonhydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases fromClostridium perfringens andArthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.Abbreviations Cer ceramide - GM3 Neu5Ac(2–3)Gal(1–4)Glc(1-1)Cer - GM4 Neu5Ac(2–3)Gal(1-1)Cer Gangliosides were abbreviated according to Svennerholm [1] and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature [2].     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

Sialyltransferases of developing rat brain

The activity of four different sialyltransferases acting on N- or O-linked chains of glycoproteins was studied in brains of 19 days-old embryos, 1 day-old newborns and adult rats. By using asialofetuin, fetuin andN-acetyllactosamine as acceptors, it has been possible to measure independently the following enzyme activities: CMP-NeuAc:Gal1-3GalNac (2–3)-sialyltransferase (EC 2.4.99.4), CMP-NeuAc:Gal1-4GlcNAc (2–3)-sialyltransferase (EC 2.4.99.6), CMP-NeuAc:Gal1-4GlcNAc (2–6)-sialyltransferase (EC 2.4.99.1) and CMP-NeuAc:NeuAc2-3Gal1-3GalNac (2–6)-sialyltransferase (EC 2.4.99.7). The specific activity of the first three enzymes which act on asialylated acceptors showed a 2.6-fold decrease in a parallel manner after ontogenic development, while the activity of NeuAc2-3Gal1-3GalNac (2–6)-sialyltransferase was four times lower in adult than in embryonic brain, showing a stronger dependence on ontogenic development. Despite the higher level of sialyltransferases able to act on glycoproteins, in fetal brain these glycoproteins do not contain a higher amount of sialic acid.Abbreviations HPLC high performance liquid chromatography - N-CAM neural cell adhesion molecule     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.     

Photoinhibition of photosynthesis under natural conditions in ivy (Hedera helix L.) growing in an understory of deciduous trees

We investigated to what extent south-exposed leaves (E-leaves) of the evergreen ivy (Hedera helix L.) growing in the shadow of two deciduous trees suffered from photoinhibition of photosynthesis when leaf-shedding started in autumn. Since air temperatures drop concomitantly with increase in light levels, changes in photosynthetic parameters (apparent quantum yield, _i and maximal photosynthetic capacity of O₂ evolution, P_max; chlorophyll-a fluorescence at room temperature) as well as pigment composition were compared with those in north-exposed leaves of the same clone (N-leaves; photosynthetic photon flux density PPFD< 100 mol · m^–2 · s^–2) and phenotypic sun leaves (S-leaves; PPFD up to 2000 mol · m^–2 · s^–1).In leaves exposed to drastic light changes during winter (E-leaves) strong photoinhibition of photosynthesis could be observed as soon as the incident PPFD increased in autumn. In contrast, in N-leaves the ratio of variable fluorescence to maximum fluorescence (F_V/F_Mm) and _i did not decline appreciably prior to severe frosts (up to -12° C) in January. At this time, _i was reduced to a similar extent in all leaves, from about 0.073 mol O₂ · mol^–1 photons before stress to about 0.020. Changes in _i were linearly correlated with changes in f_v/f_m (r = 0.955). The strong reduction in F_V/F_M on exposure to stress was caused by quenching in F_M. The initial fluorescence (F₀), however, was also quenched in all leaves. The diminished fluorescence yield was accompanied by an increase in zeaxanthin content. These effects indicate that winter stress in ivy primarily induces an increase in non-radiative energy-dissipation followed by photoinhibitory damage of PSII. Although a pronounced photooxidative bleaching of chloroplast pigments occurred in January (especially in E-leaves), photosynthetic parameters recovered completely in spring. Thus, the reduction in potential photosynthetic yield in winter may be up to three times greater in leaves subjected to increasing light levels than in leaves not exposed to a changing light environment.Abbreviations and Symbols F₀, F_M initial and maximal fluorescence yield when all PSII centres are open and closed - F_V variable fluorescence (F_M-F₀) - P_max maximal photosynthetic capacity at 1000 umol · m^–2 · s^–1 PPFD and CO₂ saturation - PPFD photosynthetic photon flux density - _i apparent quantum yield of photosynthetic O₂ evolution - E-leaves, N-leaves shade leaves exposed, not exposed to drastic light changes during winter - S-leaves sun leaves from an open ivy stand Dedicated to Professor Otto Härtel on the occasion of his 80th birthdayThis work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung.     

RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley

Summary To identify the mildew resistance locus Mla in barley with molecular markers, closely linked genomic RFLP clones were selected with the help of near-isogenic lines having the Pallas and Siri background. Out of 22 polymorphic clones 3 were located around the Mla locus on chromosome 5 with a distance of 5.1 + 2.9 cM (MWG 1H068), 4.2±1.7 cM (MWG 1H060) and 0.7 ± 0.7 cM (MWG 1H036), respectively. The polymorphic clone MWG 1H036 displayed the same RFLP pattern in both Pallas and Siri near-isogenic lines and in different varieties digested with six restriction enzymes possessing the same mildew resistance gene. The alleles of the Mla locus were grouped in 11 classes according to their specific RFLP patterns; 3 of these groups contain the majority of Mla alleles already used in barley breeding programs in Europe.     

Improved growth of the red yeast, Phaffia rhodozyma (Xanthophyllomyces dendrorhous), in the presence of tricarboxylic acid cycle intermediates

Catabolites related to tricarboxylic acid cycle affected growth and carotenogenesis in Phaffia rhodozyma. Glutamate, glutamine, aspartate, asparagine and proline at 75 mM of N increased biomass from 2 g l^–1 to 2.9–4.7 g l^–1 but decreased carotenoid from 420 g g^–1 yeast to 200–260 g g^–1 yeast in strain 67-385. However, simple nitrogen sources did not decrease carotenoid formation. Tricarboxylic acid intermediates repressed carotenogenesis to a less degree than the corresponding amino acids. Carotenoid hyper-producing mutants were impaired in nitrogen utilization. These results indicated that nitrogen assimilation and the concentrations of tricarboxylic acid cycle intermediates are involved in regulation of carotenoid biosynthesis.     

Effect of gramine on the feeding behavior of the aphids Schizaphis graminum and Rhopalosiphum padi

The effects of the indole alkaloid gramine on the behavior of the aphids Schizaphis graminum and Rhopalosiphum padi feeding on barley seedlings and on artificial diets were studied. On barley cv. F. Union, which lacks gramine, S. graminum ingested from phloem tissue for 30–80 min and non-phloem for 20–40 min, over a period of 3 h. In cultivar Datil S, gramine was found only in the epidermis and parenchyma mesophyll cells. On this cultivar, the non-phloem wave form of S. graminum was not observed. On F. Union, R. padi ingested non-phloem with short periods of ingestion from phloem. On cv. Datil S, this aphid did not show the non-phloem wave form. In experiments performed with cv. F. Union seedlings that contained exogenous gramine S. graminum did not ingest from phloem. Exogenous gramine was found only in the vascular bundles. It is suggested that gramine content and location may affect the feeding behavior of aphids in barley.

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Résumé Les effects de la gramine sur le comportement alimentaire des pucerons Schizaphis graminum et Rhopalosiphum padi on été étudiés en plantules d'orge (Hordeum distichum) et aussi avec des diètes artificielles à l'aide d'un moniteur électronique. S.graminum se nourri pendant 30–80 minutes à partir du phloème et il a pris 20–40 minutes en se nourrisant d'autres tissues divers, d'un total de 3 heures d'exposition à la plante. Dans la culture Datil S la gramine a été trouvée seulement dans les cellules de l'epidermis et du parenchyme du mesophyle. Dans ce cas, l'onde non-phloematique de S. graminum, n'a été pas observé; le temps pour le première onde X a été plus prolongé par rapport à la culture F. Union. Dans la culture F. Union, R. padi se nourri principalement de tissue non-phloematique (35–100 minutes) et du phloème par des brèves périodes (15–25 minutes). Dans le cas de la culture Datil S ce puceron n'a pas montré l'onde X. D'autre part, quand les plantules de F. Union ont été poussées dans des solution avec gramine, S. graminum ne se nourri pas du phloeme, tandis que R. padi montre un comportement similaire a celui-ce observé avec plantules sans gramine. La gramine absorbé par les plantules a été retrouvée seulement dans les faisceaux vasculaires. D'autre part, la gramine dans des diètes artificielles agit comme repulsif alimentaire à partir de concentrations de 0,5 mM. Avec des diètes contenant 3 mM de gramine l'alimentation des pucerons a été inhibée. Pourtant, ces resultats sugèrent que le contenuet et la localisation de la gramine peuvent modifier le comportement alimentaire des pucerons en plantules d'orge.

     

Storage,mobilization and interrelations of starch,sugars, protein and fat in the ray storage tissue of poplar trees

The seasonal pattern in starch, various sugars, protein, and fat, and their interrelationship, has been followed in 3-year-old branch wood of poplar trees (Populus x canadensis Moench robusta) under natural site conditions. The deposition of starch, protein and fat proceeds at different times. Starch accumulates from May until October, fat mainly during the summer months, and protein when the leaves are yellowing in September and October. The maximum concentrations in the branch wood were 15–18 g starch, 6–9 g protein, 4–8 g fat, 10–15 g sucrose, and up to 30 g total sugars per milligram dry weight (DW). During starch deposition periods no increased sucrose level is found in the tissue. The maximum daily starch deposition rate was 0.2–0.4 g starch/day/mg DW of wood. During starch hydrolysis in late autumn and winter, a dramatic increase in sucrose and its galactosides is measured (up to 15–27 g/mg DW in total). In early spring, before budbreak, the concentrations of these sugars diminishes sharply. In contrast to this clear-cut starch-to-sugar conversion in autumn no significant starch-to-fat conversion is detected. An elevated content of free glycerol, however, is found in winter. In spring, starch and storage protein are mobilized completely, or almost completely, in poplar twig wood. A noteworthy pool of maltose is found transiently during autumn (up to 8 g/mg DW) and again in spring. The results demonstrate that the individual storage materials, e.g. starch, protein, and fat, are accumulated fairly independently in the wood storage parenchyma. Tissue sugar levels, in contrast, appear to be closely related to the seasonal variations in starch content, on the one hand, and to the acclimation and deacclimation of the cells, on the other. The interrelations of the storage materials and sugars are discussed.     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose     

Improvement on laboratory fermentation equipment to study Saccharomyces cerevisiae metabolism

Beside being an ordinary fermenter, the present equipment was conceived to sample the medium, to store the samples and to record photographs of the yeasts. Ten sensors were used to measure gas exchanges. During the growth of ScM1 (a Saccharomyces cerevisiae strain) on glucose, we could observe two different linear decreases of CO₂ production rates (18.17±0.12 mmol CO₂ h^–2 (g biomass)^–1 and 8.67±0.12 mmol CO₂ h^–2 (g biomass)^–1), together with a sudden variation of slope during the respiro-fermentative phase. Nomenclature F_in InletairFlowl h ^–1 F_out OutletgasFlowl h ^–1 _in Inletairtemperature°C_out Outletgastemperature°CP _atm AtmosphericPressuremmHgP _in InletairOverPressuremmHgP _out OutletgasOverPressuremmHgDODissolvedO ₂ mg l^–1 pO₂ PartialPressureO ₂ in Outlet gas % (v/v) pCO₂ PartialPressureCO ₂ in Outlet gas % (v/v) Int(t) Whole number of hours     

Comparison of laboratory- and field-derived soil water retention curves for a fine sand soil using tensiometric,resistance and capacitance methods

The approximate range from 100 to 50% of plant-available water in Apopka fine sand (loamy, siliceous, hyperthermic Grossarenic Paleudult) is 0.08–0.04 cm³ cm^–3 soil water content () or –5 to –15 kPa of soil water matric potential (). This narrow range of plant-available soil water is extremely dry for most soil water sensors. Knowledge of the soil water retention curves for these soils is important for effective irrigation of crops in fine sand soils of subtropical and tropical regions of the world. The primary objective of this study was to compare sandy soil water retention curves in the field as measured by tensiometer and resistance block values and capacitance sensor . The second objective was to compare these curves to one developed on a Florida fine sand soil using a pressure plate apparatus. Tensiometer and resistance block values were compared to values from capacitance sensors calibrated gravimetrically. The effective range of both tensiometers and resistance sensors in fine sand soils is between –5 and –20 kPa . Soil water potential values for both sensors were within 2 kPa of the mean for each sensor. Change in was similar over the range of 0.04–0.08 cm³ cm^–3 . Curves for the two sensors were different by 4 kPa at 0.04 cm³ cm^–3. The relationship between and were similar at 10–20, 20–30 and 40–50 cm depths. This was not true for a laboratory determined soil water retention curve for the same soil type. These differences are significant in soils with very low water holding capacities. Differences between laboratory- and field-determined retention curves could be due to a combination of entrapped air in the field soil and/or alteration in bulk density in the laboratory samples.     

Systemic induction of volatile release in cotton: How specific is the signal to herbivory?

Plants attacked by herbivorous insects release chemical signals that attract natural enemies of the herbivores to the damaged plants. Feeding of Spodoptera exigua larvae on the lower leaves of cotton (Gossypium hirsutum L.) for multiple feeding periods of 9–12 h with a 12 h, interval in between when the caterpillars are removed overnight, will induce a systemic release of volatile compounds that is comparable to the volatiles released in response to continuous feeding damage on the lower leaves for several days. The systemic volatile release in response to herbivory can be mimicked by mechanically damaging the lower leaves and applying caterpillar oral secretion to the injured leaves over 4 days. Cotton plants that are only mechanically damaged systemically release significantly less -pinene, myrcene, (Z)-3-hexenyl acetate, (E)--farnesene and (E,E)--farnesene after 4 days compared to plants damaged mechanically with application of caterpillar regurgitant. However, multiple 9–12 h mechanical damage alone induces a significantly higher systemic release of (Z)-3-hexenyl acetate, myrcene, (E)--ocimene, and (E)--farnesene after 4 days compared to undamaged control plants. This indicates that multiple mechanical damage alone cannot mimic completely the response induced by mechanically injuring the leaves and applying caterpillar regurgitant. A specific elicitor in the regurgitant of the caterpillar enhances the amount of several systemically released volatiles. Thus, the systemic release of volatile compounds by herbivore-damaged cotton plants appears to be regulated by at least two different mechanisms.     

Purification of UDP-galactose: diacylglycerol galactosyltransferase from chloroplast envelopes of spinach (Spinacia oleracea L.)

Uridine 5-diphosphate(UDP)-galactose: 1,2-diacylglycerol 3-O--d-galactopyranosyltransferase (EC 2.4.1.46) is an integral protein of chloroplast envelope membranes from which it has been partially purified (Covès et al., 1986, FEBS Lett. 208, 401–406). We have worked out a purification procedure which after removal of peripheral membrane proteins, solubilization and two chromotographic steps allowed us to identify a 22-kDa protein as the galactosyltransferase. Enrichment of enzymatic activity was paralleled by an enrichment of this protein and its radioactive derivative obtained by photoaffinity labelling with [-^–32P]UDP which is a potent inhibitor of the enzyme. The purification factor of about 350 is substantially higher than achieved previously and indicates that the enzyme represents less than 0.3% of the envelope proteins. The purified enzyme has a Km of 87 M for UDP-galactose with dioleoylglycerol as acceptor and could not be activated by addition of other lipids.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate - DTE dithioerythritol - MGD monogalactosyl diacylglycerol - PMSF phenylmethanesulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by the Deutsche Forschungsgemeinschaft.     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

Synthesis of optically pure chrysobactin and immunoassay development

Chrysobactin (-N-(2,3-dihydroxybenzoyl)-d-lysyl-l-serine), a siderophore that is essential for systemic virulence by plant pathogenic Erwinia chrysanthemi, was synthesized with high diastereomeric purity. Chrysobactin was prepared by coupling the N-hydroxysuccinimide ester of -N-(2,3-dibenzyloxybenzoyl)--N-Cbz-d-lysine with l-serine benzyl ester followed by deprotection via hydrogenolysis. Optically pure chrysobactin was obtained with 98% overall yield. A monoclonal antibody to ferric chrysobactin was developed and characterized as IgM. The antibody reacts with chrysobactin, ferric chrysobactin and less strongly with ferric dihydroxybenzoic acid. The antibody reacts weakly with the siderophores ferrichrome, A, ferric pseudobactin and ferric rhodotorulic acid. This antibody was used in a competitive immunoassay to detect ferric chrysobactin at 10^–8 to 10^–10 mol. This immunoassay may provide a useful method for the detection of chrysobactin in plant samples.