Effects of different sterols on the inhibition of cell culture growth caused by the growth retardant tetcyclacis

Cell division in cell suspension cultures can be completely blocked by the growth retardant tetcyclacis at a concentration of 10^-4 mol l^-1. In rice cells it has been demonstrated that the growth inhibition can be completely overcome by application of cholesterol independent of the duration of pretreatment with tetcyclacis. In suspension cultures of maize and soybean, too, the effect of tetcyclacis on cell division was neutralized by adding cholesterol. Other plant sterols, stigmasterol, campesterol and sitosterol were active in a decreasing order. Modifications in the cholesterol perhydro-cyclopentanophenanthrene-ring system indicate that the hydroxyl group at C-3 and the double bond between C-5 and C-6 in ring B are required for the activity. In contrast, gibberellic acid as well as ent-kaurenoic acid could not compensate retardant effects. Likewise, tetcyclasis did not change the level of gibberellins in rice cells as shown by radioimmunoassay. Thus, it is concluded that in cell suspension cultures sterols play a more important role in cell division than gibberellins.Abbreviation GA_x gibberelin A_x     

Comparison of gas chromatography-mass spectrometry,radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.)

Quantitative estimates of gibberellin A₉ in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A₉, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.Abbreviations FW fresh weight - GA₉ gibberellin A₉ - GA₉–Me methylated GA₉ - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MID multiple-ion detection - RIA radioimmunoassay     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.     

Putative neurotransmitters in the retinae of three urodele species (Triturus alpestris,Salamandra salamandra,Pleurodeles waltli)

Summary The immunocytochemical localization of several substances with putative neurotransmitter or modulator properties was investigated in the retinae of three urodele species. Gamma-aminobutyric acid-like immunoreactive labelling appeared in different types of amacrine and horizontal cells. In addition, labelled fibres in the optic nerve were detected. It was not possible to determine whether these fibres were ganglion-cell axons or part of an efferent projection. Endogenous serotonin was found in several populations of amacrine cells including stratified and diffuse types. Glucagon-like immunoreactivity appeared in one bistratified amacrine cell type, and neurotensin-like immunoreactivity was detected in a single monostratified amacrine cell type. Metenkephalin-like-immunoreactive labelling was rare but found in several sublaminae of the inner plexiform layer. Thus each peptide-like-immunoreactive cell type makes up a distinct and unique population of cells and probably has a special functional role in retinal processing. There are striking similarities in the peptide-like immunoreactive patterns of Triturus alpestris and Necturus maculosus whereas in Ambystomatidae the peptide-like-immunoreactive systems appear to be differently organized. This supports the hypothesis that Salamandridae and Proteidae are more closely related to each other than to the Ambystomatidae.Abbreviations GABA gamma-aminobutyric acid - GCL ganglion cell layer - Glu glucagon - HRP horseradish peroxidase - INL inner nuclear layer - IPL inner plexiform layer - IR immunoreactive or immunoreactivity - M-enk metenkephalin - Neu neurotensin - OFL optic fibre layer - ONL outer nuclear layer - OPL outer plexiform layer - Ser serotonin This work forms part of the doctoral thesis of Gaby Gläsener, Faculty of Biology, Technical University of Darmstadt, Federal Republic of Germany. Supported by a research grant from the Deutsche Forschungsgemeinschaft (Hi 306/1-1)     

Methyljasmonate and α-linolenic acid are potent inducers of tendril coiling

A coiling-inducing factor was isolated from tendrils of Bryonia dioica Jacq. and identified by infrared, ¹H-, ¹³C-nuclear magnetic resonance and mass spectrometry as -linolenic acid. When applied to detached tendrils, exogenous -linolenic acid, but not linoleic acid or oleic acid, induced tendril coiling. Further investigations showed that metabolites of -linolenic acid, jasmonic acid and, even more so, methyljasmonate, are highly effective inducers of tendril coiling in B. dioica. Methyljasmonate was most active when administered by air and, in atmospheric concentrations as low as 40–80 nM, induced a full free-coiling response with kinetics similar to mechanical stimulation. Even atmospheric levels as low as 4–5 nM methyljasmonate were still found to be significantly active. Methyljasmonate could be one of the endogenous chemical signals produced in mechanically stimulated parts of a tendril and, being highly volatile, act as a diffusible gaseous mediator spreading through the intracellular spaces to trigger free coiling of tendrils.Abbreviations EI-MS electron impact-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - NMR nuclear magnetic resonance - TFA trifluoroacetic acid We are indebted to the Deutsche Forschungsgemeinschaft, Bonn and the Fonds der Chemischen Industrie, Frankfurt (literature provision) for their support and to Dr. C. Brückner, Halle, for jasmonic-acid determinations.     

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.     

Monoclonal antibodies to plant growth regulators. II. Indole-3-acetic acid

The production and characterization of high-affinity monoclonal antibodies suitable for the radio- and enzymeimmunoassay of the endogenous plant growth regulator, indole-3-acetic acid (IAA), is reported. Hybridomas were produced by fusion of NS 1 myeloma cells with spleen cells from Balb/c mice immunized with IAA-bovine serum albumin conjugates. From an initial collection of 158 wells containing cells secreting monoclonal antibodies against IAA, seven were used to derive cell clones. Three of these are described here. They secrete immunoglobulin (IgG2a or IgG2b) of high affinity and specificity for IAA methyl ester and can be used to quantite picogram amounts of this compound in plant extracts by radio- and enzymeimmunoassay.     

Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L

The protein(s) that constitute(s) the ATP-driven Ca²⁺-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca²⁺-transport system was studied using ATP-driven ⁴⁵Ca²⁺ import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent K_m for Ca²⁺ (5.2 micromolar), inhibition by relatively high levels of vanadate (IC₅₀ = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC₅₀ = 0.01 micromolar) and had a low apparent K_m for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca²⁺-transport detectably in the reconstituted system. However, low levels of the Ca²⁺-ionophore A 23187 instantaneously discharged 90% of the Ca²⁺ associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca²⁺-transport in the reconstituted system was thus primary active, through a Ca²⁺-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca²⁺-ATPase and for studying structural and functional aspects of the purified enzyme.