A carbene-generating photoaffinity probe for beta-adrenergic receptors.

A new radioiodinated (2.2 Ci/mumol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [125I]ICYP-diazirine binds with high affinity (Kd = 60 pM) to beta-receptors from turkey erythrocyte membranes. Upon irradiation, [125I]ICYP-diazirine is covalently incorporated in a Mr 40 000 protein. Stereoselective inhibition of photolabeling by the (-)enantiomers of alprenolol and isoproterenol indicated that the Mr 40 000 protein contains a beta-adrenergic binding site. The yield of specific labeling was up to 8.2% of total beta-receptor binding sites. The Mr 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [125I]ICYP-diazirine resulted in specific labeling of two proteins with Mr 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximately Mr 67 000) was labeled specifically.

The use of filter hybridization techniques for the identification, differentiation and classification of plant viruses.

In attempts to use dot-blot hybridization tests for the identification of viruses or for assigning them to a certain taxonomic group we found that hybridization signals may be given not only by the homologous virus, but also by heterologous viruses belonging to the same or different taxonomic groups. Possible reasons for this phenomenon, which was observed with uncloned as well as with cloned cDNAs, are discussed. Quantitative dot-blot hybridization tests with extracted viral RNAs proved to be very sensitive in differentiating closely related viruses which were barely distinguishable in serological tests. Estimates on the degree of homology between the RNAs of different viruses may be influenced by a number of experimental parameters, such as competition for the available cDNA between homologous and heterologous RNAs or homologous RNAs in different concentrations on the same sheet of nitrocellulose, saturation phenomena due to close packaging of highly concentrated RNA on the blot and, of course, stringency conditions during washing procedures. Taking these parameters into account we have reestimated the degree of homology between the RNAs of 5 tombusviruses. Our new data suggest that the order of sequence for the relationships among these 5 tombusviruses is similar to that proposed by Koenig and Gibbs (1986) on the basis of serological data.

Zygocactus virus X-based expression vectors and formation of rod-shaped virus-like particles in plants by the expressed coat proteins of Beet necrotic yellow vein virus and Soil-borne cereal mosaic virus.

Expression vectors were constructed from 35S promoter-containing full-length cDNA clones of Zygocactus virus X (ZVX). The expression of foreign genes was driven by the ZVX coat protein (cp) subgenomic promoter. It was successful only when the variable region downstream of the conserved putative promoter region GSTTAAGTT(X(12-13))GAA was retained. Most of the ZVX cp gene, except for a short 3' part, was replaced by the corresponding sequence of the related Schlumbergera virus X (SVX) and its cp subgenomic promoter to enable encapsidation of the transcribed RNA by an SVX/ZVX hybrid cp. Vector-expressed cp of Beet necrotic yellow vein virus (BNYVV) assembled in Chenopodium quinoa, Tetragonia expansa and Beta vulgaris leaves into particles resembling true BNYVV particles. The virus produced from these constructs retained its ability to express BNYVV cp in local infections during successive passages on C. quinoa. This ability was lost, however, in the rarely occurring systemic infections.

[Standardized forced diuresis and peritoneal dialysis in the therapy of organophosphorous poisoning (author's transl)]. / Standardisierte forcierte Diurese und Peritonealdialyse in der Behandlung einer Alkylphosphatvergiftung.

In a case of severe poisoning with the organophosphorous insecticide methidathione standardized forced diuresis and peritoneal dialysis were performed for the first time in addition to the usual therapeutical procedures (e.g. repeated gastric lavage, induced diarrhea, atropine and intensive care). The course of the illness as well as the duration of the acute phase were significantly shorter than in comparable cases. No hazardous situation occurred after the first hours of treatment. The presence of the organophosphorous cholinesterase inhibitor or of its metabolites in the urine and the dialysate was proved by thin-layer chromatography.

Prostaglandin E1 action on adenylate cyclase activity and membrane lipids in platelets.

The effect of prostaglandin E1 (PGE1) on temperature-induced membrane lipid phase transitions of human blood platelets was investigated using excimer forming 1-pyrenedecanoic acid as fluorescent probe. PGE1 and PGF2 alpha at 10 microM concentrations had a small but reproducible effect on the melting curves between 20 degrees and 30 degrees C and lowered the temperature for the onset of the phase transition from 26 degrees to 21 degrees C. The adenylate cyclase of the platelet membrane was half maximally stimulated by PGE1 at 0.8 microM and maximally activated at 10 microM concentrations. PGF2 alpha at 100 microM concentrations produced only 40% of the maximal stimulation achieved with PGE1. Stimulation of adenylate cyclase by 10 microM PGE1 increased steeply with temperature between 20 degrees to 30 degrees C. Binding experiments with [3H]PGE1 indicated a high affinity and a low affinity site with KD 13nM and 2 microM, respectively. Non-radioactive PGE1 inhibited binding of [3H]PGE1 to both sites at concentrations corresponding to their respective dissociation constants. PGF2 alpha inhibited binding of PGE1 only at greater than or equal to 1 microM concentrations. It is assumed that only the high affinity binding sites represent specific PGE1 receptors. Binding of PGE1 to the low affinity sites and the observed effects on membrane structure may result from interactions of PGE1 with membrane lipids. Since these latter effects and also stimulation of adenylate cyclase required similar concentrations, it is proposed that local interactions of PGE1 with membrane lipids are a prerequisite for specific receptor-mediated adenylate cyclase activation.

Photoaffinity labeling of the beta-adrenergic receptor with azide derivatives of iodoccyanopindolol.

Two photosensitive iodocyanopindolol derivatives, 1-(4-azidobenzimidyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-1) and 1-(4-azidobenzoyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-2) have been prepared. [125I]ICYP-azide-1 and -2 (specific radioactivity up to 2.2 Ci/mumol) bind specifically and with very high affinity (KD = 40-45 pM) to beta-adrenergic receptors of turkey erythrocyte membranes. When [125I]ICYP-azide-1 or -2 were incubated with membranes and UV-irradiated, two polypeptides (Mr = 40,000 and 50,000) were specifically photolabeled as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides may represent subunits of the beta-adrenergic receptor. The yield of specific covalent label incorporation into both polypeptides was up to 17.2% with [125I]ICYP-azide-2 when expressed as fraction of total beta-receptor binding sites. Since the Mr = 40,000 polypeptide was labeled predominantly and since covalent incorporation had the same concentration dependence as reversible specific binding, this polypeptide could contain a beta-adrenergic ligand binding site. Due to the low working concentration (10-100 pM) of [125I]ICYP-azide-1 and -2, nonspecific labeling of membrane proteins was extremely low. The new photoaffinity labels should therefore become valuable tools for probing beta-receptor structure.

Histrionicotoxin enhances agonist-induced desensitization of acetylcholine receptor.

Dihydroisohistrionicotoxin inhibits acetylcholine receptor-dependent 22Na+ uptake of cultured chick muscle cells with a KI of 0.2 micrometer. The inhibition is noncompetitive with respect to agonists. The toxin enhances desensitization of the receptor by agonists which is accompanied by a 10-fold increase in receptor affinity for agonists. Dihydroisohistrionicotoxin increases the affinity of the desensitized form of the receptor for agonists but not antagonists. The results suggest that dihydroisohistrionicotoxin inhibits the acetylcholine receptor by causing an increase in the affinity of the desensitized form of the receptor for agonists and thereby stabilizing the desensitized state.

Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana.

The coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli. For expression in higher plants the scFv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from the constructs. The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants. The scFv were not secreted into the culture fluids of suspension cultures, but were retained in the cells. The amount of expression of scFv in the best expressing plants was at least as high as in bacterial culture supernatants. In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E. coli or from plants. The majority of the 21 plants expressing cp-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots.

Molecular characterisation of potexviruses isolated from three different genera in the family Cactaceae.

The genome properties of three potexviruses which previously had been isolated from different genera in the family Cactaceae and had been found to be only distantly related serologically have been studied. The sequence of the 3040 3' terminal nucleotides of the genomic RNA of isolate K11 from Schlumbergera bridgesii and the complete RNA sequences of isolates B1 and CC10 from Zygocactus sp. and Opuntia sp., respectively, were determined. Starting sequences were obtained by means of immunocapture reverse transcription PCR using primers derived from highly conserved sequences in other potexviral RNAs. The known parts of the sequences were extended by means of random-primed cDNAs and specific primers derived from the known parts of the sequences. The genome structure of the three viruses resembles that of other potexviruses. The conserved motifs typical for replication-associated proteins, triple gene block (TGB) proteins and coat proteins of potexviruses were readily identified in the translation products of the five open reading frames. The 3' untranslated regions of the three RNAs are folded into secondary structures containing three characteristic hairpins. Rather low percentages of amino acid sequence identities ranging from 62% to 76% for the coat proteins and 41% to 49% for TGB proteins 3 suggest that these viruses should be regarded as distinct virus species for which the names Zygocactus virus X, Schlumbergera virus X and Opuntia virus X are proposed. It is also suggested that the name Cactus virus X which originally was coined for all three virus isolates should no longer be used.

Effect of recombinant beet necrotic yellow vein virus with different RNA compositions on mechanically inoculated sugarbeets.

Beet necrotic yellow vein virus (BNYVV) inocula with different RNA compositions were prepared from infectious transcripts of RNAs 3 and 4 and the Rg 1 isolate, which has a genome consisting only of RNAs 1 and 2. The recombinant viruses were inoculated on 6- to 8-day-old sugarbeet seedlings by 'vortexing'. Inocula containing RNAs 1 and 2 or 1, 2 and 4 produced some growth reduction, but the most dramatic effects, with yield reductions of about 95% in a highly susceptible variety, were seen when RNA 3 was also present in the inoculum. Under these conditions the side roots were brown and brittle and often deteriorated, but 'root beardedness' was not observed. This might be due to the fact that our experiments were done in the absence of Polymyxa betae. Alternatively, the heavy inoculation at a very young age may either have weakened the plants to such an extent that extensive root proliferation was impaired or it may have led to rapid deterioration of the proliferating rootlets, which would therefore be lost prior to or during removal of the tap roots from the soil. In the presence of RNA 3 the virus concentrations in tap roots were markedly increased suggesting that this RNA facilitates the multiplication and/or spread of the virus in root tissues.

cDNAs of beet necrotic yellow vein virus RNAs 3 and 4 are rendered biologically active in a plasmid containing the cauliflower mosaic virus 35S promoter.

cDNAs of beet necrotic yellow vein virus RNAs 3 and 4 could be rendered biologically active when they were placed under the control of the cauliflower mosaic virus 35S promoter and polyadenylation signal. Although the 35S in vivo transcripts should have contained up to forty 5' and several hundred 3' nonviral nucleotides, the progeny viral RNAs had the same sizes as in naturally infected sugarbeets. The progeny RNAs did not hybridize with the nonviral sequences indicating that they were apparently not replicated. Deletion and insertion mutants of RNA 3 cDNA clones were also biologically active in plants but a plasmid which contained the cDNA of RNA 3 in antisense orientation was not. The biological activity of plasmid DNAs compared with the corresponding synthetic transcripts is discussed.

Cloning of the coat protein gene from beet necrotic yellow vein virus and its expression in sugar beet hairy roots.

Expression of the beet necrotic yellow vein virus (BNYVV) coat protein (CP) gene in transgenic sugar beet hairy roots was accomplished as a step towards CP-mediated virus resistance. A cDNA for the CP gene and its 5' terminal untranslated leader sequence was prepared from BNYVV RNA, using two oligodeoxynucleotides to prime the synthesis of both strands. Second-strand synthesis and amplification of the cDNA were done by Taq DNA polymerase chain reactions. Run-off transcripts of the cloned cDNA sequence were obtained and translated in vitro, yielding immunoreactive CP. A binary vector construction containing the CP gene under the control of the 35S promoter of cauliflower mosaic virus was prepared and used for Agrobacterium rhizogenes-mediated transformation of sugar beet tissue. Stable integration and expression of the CP gene in sugar beet hairy roots was demonstrated by Southern, Northern, and Western blot analysis, respectively.

Conformations of the li-antamanide complex and na-[phe, val]antamanide complex in the crystalline state.

Antamanide, a cyclic decapeptide isolated from the poisonous mushroom Amanita phalloides, preferably complexes with Na(+), but in less polar solvents, e.g., acetonitrile, also with Li(+) or K(+). The selectivity of complexation makes it an important model for the study of conformational requirements of ion binding. The conformations of the lithium antamanide complex and the Na-[Phe(4), Val(6)]antamanide complex have been established by x-ray diffraction analyses of single crystals. The two compounds are isostructural, but not isomorphous. The complexes are folded into a globular shape with an approximate 2-fold axis. Two of the peptide linkages are in the cis conformation, Pro(2)-Pro(3) and Pro(7)-Pro(8). There are only two intramolecular hydrogen bonds. Four C==O groups have their O atoms directed inward to form four Li-O or Na-O ligands. The fifth ligand to the metal ion is provided by a solvent molecule. The conformation found in the crystalline state is different from any of the conformations proposed for the sodium antamanide complex in solution on the basis of nuclear magnetic resonance data.

Conformation of uncomplexed [Phe4, Val6] antamanide crystallized from nonpolar solvents.

[Phe4, Val6] antamanide, a synthetic, biologically active analog of the cyclic decapeptide antitoxin isolated from Amanita phalloides, has been crystallized from a mixture of n-hexane and methyl acetate, and its conformation has been established by the direct method of x-ray diffraction analysis, i.e., without the benefit of any heavy atom. The uncomplexed molecule contains a 2-fold rotation axis and cis peptide linkages between Pro2-Pro3 and Pro7-Pro8. Otherwise, its conformation differs extensively from that of the Na+[Phe4, Val6] antamanide-C2H5OH complex. The 30-membered ring is elongated and relatively planar as compared to the folded ring in the Na+ complex. The six NH groups are directed toward the anterior of the molecule. There is one pair of intra-molecular NH---O=C bonds of the 5 leads to 1 type containing a cis peptide unit. The other four NH groups participate in hydrogen bonds to three H2O sites in the interior of the molecule. The 10 hydrophobic side groups cover the bottom and surround the perimeter of the molecule with the phenyl groups in the four Phe residues folded against the molecule. The conformation found for [Phe4, Val6] antamanide crystallized from nonpolar solvents is different from any conformations proposed for antamanide in solution based on nuclear magnetic resonance data.

Restriction fragment length polymorphism analysis of reverse transcription-PCR products reveals the existence of two major strain groups of beet necrotic yellow vein virus.

Beet necrotic yellow vein virus (BNYVV)-infected sugarbeets were obtained from many parts of Europe and also from some sites in Asia and the U.S.A. Reverse transcription (RT)-PCR products of more than 1 kbp were obtained for four different regions of the viral genome which may be particularly important with respect to the pathogenic properties of the virus, i.e. for the coat protein and the 42K protein-encoding regions on RNA 2 and for major parts of RNAs 3 and 4. Restriction fragment length polymorphism (RFLP) patterns obtained with these PCR products revealed the existence of two major strain groups of BNYVV, named type A and type B. The A type was detected in Greece, the former Yugoslavia, Slovakia, parts of Austria, Italy, Spain, parts of France, Belgium, The Netherlands and England as well as in Asia (Turkey, Kazachstan, China and Japan) and the U.S.A. The B type occurs in Germany and parts of France. Mixed infections were detected at the borderline regions between areas of the A and B types. Comparisons of published and newly determined nucleotide sequences of the respective parts of the BNYVV genome indicate that the percentage of nucleotide differences between the A and the B type is approximately 3% for the respective regions of RNAs 2 and 3 and approximately 1.5% for RNA 4. Nucleotide sequences appear to be remarkably stable within each of the two strain groups. The majority of the nucleotide differences between the A and B types occur in the third triplet position. The amino acid changes in the coat protein area are outside the four previously determined antigenic regions that are accessible on the surface of the virus particles and are involved in the formation of continuous and presumably also discontinuous epitopes. This may explain why serological differences between the two strain groups have not been found.

Antigenic analysis of the coat protein of beet necrotic yellow vein virus by means of monoclonal antibodies.

By means of monoclonal antibodies (MAbs), five (groups of) epitopes were identified on particles of beet necrotic yellow vein virus (BNYVV). Epitopes 1 and 2, which were located on the opposite extremities of virus particles, are discontinuous (SDS-labile) epitopes which were destroyed when the particles were treated with trypsin. Epitope 3 is a continuous (SDS-stable) epitope located at the same extremity as epitope 2. It was not destroyed when the particles were treated with trypsin and was present on an Escherichia coli-expressed fusion protein containing amino acids (aa) 1 to 103 of the BNYVV coat protein. The continuous epitope 4, which was located along the entire length of the particles, was found to be present on a fusion protein containing aa 104 to 188 of the BNYVV coat protein but not on trypsin-treated virus particles. In Western blots, these treated particles yielded two slightly smaller coat proteins which failed to react with MAbs specific for epitope 4 but did react with polyclonal antisera and MAbs specific for epitope 3. BNYVV coat protein has a trypsin cleavage site on the carboxyl side of arginine in position 182, so it is therefore suggested that epitope 4 is located on the exposed C terminus, which is composed of aa 183 to 188. Epitope 5 was also located along the entire length of the particles but in a more uneven distribution than epitope 4. This may be because it is a discontinuous epitope that is very sensitive to subtle changes in protein conformation.

Epitope mapping on fragments of beet necrotic yellow vein virus coat protein.

The location of five SDS-stable epitopes on the coat protein (CP) of beet necrotic yellow vein virus was determined by reacting Escherichia coli-expressed free CP, as well as fusion proteins (FP) containing fragments of the CP, with polyclonal and monoclonal antibodies on Western blots. Epitope 1, which has previously been found to be exposed on only one extremity of the virus particle, was located in the region between amino acids (aa) 1 and 7, i.e. on the N terminus of the CP. It was blocked when the N terminus of the CP was linked to a portion of the beta-galactosidase sequence in an FP. Epitope 3, which has previously been found to be exposed on the opposite extremity of the particle, was located in the region between aa 37 and 59. Epitope 4, which is exposed along the entire length of the particle, occurs on the C terminus of CP (aa 183 to 188). Two previously unknown epitopes were identified in the regions between aa 115 and 125 and 125 and 140, respectively. The former was located on the same extremity of the particle as epitope 3, the latter became accessible only after denaturation of the particle. Nothing is known about the probably non-adjacent aa sequences that participate in the formation of the two SDS-labile epitopes (epitopes 2 and 5) which are found on one extremity and along the entire length of the particle, respectively.

Perhydrohistrionicotoxin: a potential ligand for the ion conductance modulator of the acetylcholine receptor.

Histrionicotoxin from the Colombian frog Dendrobates histrionicus and its perhydro derivative reversibly block the acetylcholine-sensitive ion conductance system in frog neuromuscular preparations. The perhydro derivative and [3H]perhydrohistrionicotoxin, like histrionicotoxin, caused a significant decrease in the peak amplitude of the end-plate current and shortened its rise time and half-decay time. In membrane preparations from Torpedo electroplax, [3H]perhydrohistrionicotoxin bound reversibly to a limited number of high-affinity sites [dissociation constant, (KD) = 0.4 micronM]. The ratio of perhydrohistrionicotoxin to acetylcholine binding sites in these membrane preparations approached 2. Histrionicotoxins, local anesthetics, and certain cholinergic agonists inhibited binding of perhydrohistrionicotoxin. Binding of perhydrohistrionicotoxin to membranes was decreased by heat or treatment with proteases. Treatment of membranes with Triton X-100 solubilized acetylcholine binding proteins and apparently also perhydrohistrionicotoxin-binding proteins. However, the detergent Triton X-100 also bound [3H]perhydrohistrionicotoxin. This nonspecific binding was not saturable and complicated studies on the antagonism by drugs of binding of [3H]perhydrohistrionicotoxin. In solubilized preparations the binding protein for acetylcholine could be removed by affinity chromatography or immunoprecipitation without affecting binding of perhydrohistrionicotoxin. Sephadex chromatography also separated acetylcholine- from perhydrohistrionicotoxin-binding proteins. Perhydrohistrionicotoxin did not bind significantly to purified acetylcholine-receptor protein but presumably bound to an ion conductance modulator protein that was associated with the acetylcholine-receptor in intact membrane and readily separable from the receptor protein after solubilization.