Band 40, Heft 5-6

Cells and cell aggregates from suspension cultures are plated onto selection plates, consisting of: 1. A layer of nutrient medium solidified with agar 2. A filter paper coated on the upper side with activated charcoal (AC) as an adsorbent 3. covered by a sheet of cellophane as cell carrier. Substances produced migrate through the cellophane and are locally bound to the adsorbent layer. A fter transfer of the cells together with the cellophane to another dish the adsorbent filter is dried and the substances bound to the AC are eluted to the white side of the filter paper where they can be detected by m eans of standard methods of TLC and PC. The substance pattern mirrors the pattern of cell material and allows to discover those cells with deviating substance production. This method is used to recover cell aggregates of Nicotiana tabacum and Matricaria chamomilla from a mixture of both cultures.

Phytochemical and microscopial analysis of leaves and stems of various species of Encelia showed a strict correlation between the presence of resin ducts and the accumulation of benzopy­rans and benzofurans. Fluorescence microscopy of Encelia farinosa proved unambigously that these compounds are stored exclusivly in the resin ducts and the surrounding cells.

An electrophoretic survey of anthocyanins in flowers of 81 species belonging to 27 plant families indicated that malonated or similarly substituted zwitterionic anthocyanins occur in half the sample. In particular, almost all species surveyed in the Compositae and the Labiatae were positive. The same results were also indicated by HPLC separations. Detailed examination of the anthocyanins of Dahlia variabilis and Plectranthus argentatus confirmed the presence of malonylation through glucose. It appears that anthocyanins substituted by aliphatic dicarboxylic acids may be widespread in the angiosperms.

Isolation and identification of sterols from culture extracts of the fungus Aureobasidium (Dematium) pullulans yielded metabolites with key function in sterol synthesis. By proof of squalene, lanosterol/dihydrolanosterol, desmosterol, ergosterol, cholesterol, stigmasterol and β-sitosterol it was possible to work out a general synthesis-scheme for the building-up of sundry sterols in Ascomycetes.

A phytoalexin produced by the leaflets of seven cultivars of Arachis hypogaea (groundnut) after natural infection by Cercospora arachidicola or Phoma arachidicola has been characterised as the isoflavonoid (+)-medicarpin (3-hydroxy-9-methoxypterocarpan). Treatment of excised ground­nut leaflets with an aqueous solution of CuSO 4 or with a spore suspension of the fungus Helmin-thosporium carbonum has also been found to stimulate medicarpin biosynthesis.

Isoceroptene, a polyphenol isolated in trace amount from the farinose frond exudate of Pityro­gramma triangularis, is shown by spectroscopic means to possess the structure of one of the two possible “flavanone” isomers formed by ring closure of the “chalcone” ceroptene. The tautomeric structures best representing ceroptene and a related dihydrochalcone from Myrica gale are defined by 1 H-NMR spectroscopy.

The chemical composition of the leaf resin of Pluchea odorata has been studied from material collected in Tamaulipas, Mexico. It mostly consists of triterpenes and phytosterols and an eudesmane type sesquiterpene. Very small amounts of ten different flavonol aglycones occur, dissolved in this resin; most of them are 6-methoxy compounds. A comparison with previous results on material from El Salvador shows that the latter contains a lesser number of triterpenes and especially of flavonoids, but more sesquiterpenes, while the n-alkane composition varies only quantitatively between both populations.

Amentoflavone has been characterised from leaves of Patersonia glabrata. This is the first report of a biflavone in the Monocotyledoneae. The quinone plumbagin, a characteristic con­stituent of three dicotyledonous families, is now found to be a useful marker for the genus Aristea; it also occurs in two Sisyrinchium species and in Sparaxis tricolor. Mangiferin, a C-glucosylxan-thone known previously in the Iridaceae only from Crocus, Iris and Gynandriris has now been found in Eleutherine, Rigidella, Gelasine and Tigridia. results is discussed.

Intact spinach chloroplasts, which were isolated on a cushion of Percoll and purified on discon­tinuous sucrose gradients, were found to adsorb appreciable amounts of high specific activity, cytosolic O-methyltransferases. The latter activity was readily eliminated after washing the chloroplast pellet with 0.1 ᴍ phosphate in sucrose gradient buffer and finally with 0 .1% nonidet. Furthermore, the OMT activity of spinach leaves was resolved by chromatography on DEAE-Sepharose CL-6B into caffeic acid (COMT) and flavonoid (FOMT) O-methyltransferase ac­tivities, and purified to 30-and 50-fold, respectively. The similarities between the FOMT/COMT activity ratios and the methylation patterns of intact chloroplasts and their supernatants, as well as those of purified leaf preparations, suggest non-specific binding of cytosolic OMTs to chloroplast envelopes. It is concluded, therefore, that spinach chloroplasts are not involved in the methylation of phenylpropanoid or flavonoid compounds. These results call attention for the re-evaluation of the role of this organelle in the biosynthesis or accumulation of plant secondary metabolites.

An inhibition of growth in cress roots (Lepidium sativum L.) by irradiation with low-intensity millimeter-waves was found using a computer controlled optical system which is capable of measuring nearly continuously the length of the roots to an accuracy of ± 2 jam. The effect is reversible and, for a power density of 6 mWcm -2 , results in completely halting the root growth. It occurs within about 100 s after the onset of irradiation. The microwave-induced temperature increase at the surface of the root tip was found to be less than 0.3 °C at this power density. The effect did not show a sharp frequency dependence however it depended strongly on the polariza­tion of the microwaves with respect to the root orientation. The sensitivity of the root growth to the ambient temperature was examined. Only a weak temperature dependence was found which could not explain the observed effects. However simulating the microwave-induced temperature increase at the surface of the root tip with (incoherent) far-infrared light (λ≥20 μm) resulted in similar effects as with microwaves. Hence one can conclude that the observed effects are primarily caused by the small local irradiation induced thermal gradients across the surface of the root tip.

The chemical section deals with the synthesis of sodiumcyano- 10 B-borhydride (3) from 10 B-boric acid trimethyl ester (1) via sodium- 10 B-borhydride (2). By converting 3 with trimethylammonium chloride, trimethylamine-cyano- 10 B-borane (4) is formed which, via nitrilium tetrafluoroborate 5, is transformed by water to Trimethylamine-carboxy-dihydro- 10 B-borane (6). Trimethylamine-ethoxycarbonyl-dihydro- 10 B-borane (8b) and amine-carbamoyl-dihydro- 10 B-borane (8c) can be synthesized from the imidazolid 8a. In the biological section an orientation to the toxicity and the tumor affinity of the compounds 6, 7, and 8c are described. Further the possibility is shown by achieving a maximal concentration of 10 B-boron in the tumor whilst at the same time retaining as low a concentration of 10 B-Boron as possible in the surrounding tissue.

The hot 11 C-atom derived from (γ, n)-nuclear reaction of the 40 MeV-Bremsstrahlung of a electron linear accelerator may react with valine irradiated. Besides retention-labelling of valine new products which are essentially carrierfree may be produced. By this means leucine has been separated using HPLC and identified by electrophoresis in amounts of 25 MBq labelled com­pound.

The fatty acyl specificity of phospholipase A 1 and A 2 in homogenates of mouse bone marrow-derived macrophages was determined using phosphatidylcholine and phosphatidylethanolamine of different acylchain composition. Phosphatidylcholine with arachidonoyl at position 2 was cleaved preferentially by an alkaline phospholipase A 2 (pH-optimum 9.0) leading to selective liberation of arachidonic acid. In contrast, phosphatidylcholines with oleoyl or linoleoyl at posi­tion 2 were degraded mainly by an acid phospholipase A 1 (pH-optimum 4 -5) resulting in a conservation of these fatty acids esterified in lysophosphatides. Substrate kinetics of the alkaline phospholipase A 2 revealed a 30 fold higher affinity (K m = 3.8 x 10 -7 ᴍ) for 1-acyl-2-arachidonoyl-glycerophosphocholine compared to 1-acyl-2-oleoyl-glycerophosphocholine. The kinetic data were not influenced by endogenous lipids indicating that exogenous substrates do not equilibrate with cellular lipids. These results are suitable to explain a selective liberation of arachidonic acid from a mixture of phospholipids.

The sulfhydryl enzyme malate synthase from baker’s yeast was X-irradiated with 6 kGy in air-saturated aqueous solution (enzyme concentration: ≃ 10 mg/ml; volume: 120 μl), in the absence or presence of the specific scavengers formate, superoxide dismutase, and catalase. After X-irradiation, a small aliquot of the irradiated solutions was tested for enzymic activity while the main portion was investigated by means of small-angle X-ray scattering. Additionally, an unir­radiated sample without additives was investigated as a reference. Experiments yielded the fol­lowing results: 1. X-irradiation in the absence of the mentioned scavengers caused considerable aggregation, fragmentation, and inactivation of the enzyme. The dose D t 37 for total (= repairable + non­-repayable) inactivation resulted as 4.4 kGy. The mean radius of gyration was found to be about 13 nm. The mean degree of aggregation was obtained as 5.7, without correction for fragmenta­tion. An estimation based on the thickness factor revealed that about 19% of material might be strongly fragmented. When this amount of fragments was accordingly taken into account, a value of 7.1 was obtained as an upper limit for the mean degree of aggregation. The observed retention of the thickness factor and the finding of two different cross-section factors are in full accord with the two-dimensional aggregation model established previously (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99 - 121 (1980)). 2. The presence of catalytic amounts of superoxide dismutase and/or catalase, in the absence of formate, during X-irradiation reduced both aggregation and inactivation significantly. 3. The presence of formate (10 or 100 mᴍ) during X-irradiation led to a strong decrease of aggregation and inactivation. This effect was more pronounced with the higher formate concen­tration or when superoxide dismutase and/or catalase were simultaneously present during X-irradiation. The presence of formate also reduced the amount of fragments significantly. 4. The results clearly show that the aggregation and inactivation of malate synthase upon X-irradiation in aqueous solution are mainly caused by OH · ; to a minor extent O ·̄ 2 and H 2 O 2 are additionally involved in the damaging processes.

Ferredoxin from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity by an easy and fast procedure with a high yield (25 -30 mg/250 g wet weight of cells). An average molecular weight of 11800 was calculated from sedimentation coefficient (1.70 S) and Stokes radius (1.75 nm) data, sodium dodecyl sulfate-electrophoresis, and amino acid composition. Absorption spectrum show ed maxima at 276, 330, 420 and 460 nm in the oxidized form, with an absorption ratio (A 420 /A 276 ) of 0.54 and an extinction coefficient of 8.38 mᴍ-1· cm -1 at 420 nm. R educed ferredoxin show ed a single peak at 276 nm with shoulders at 284, 310, 390, 469 and 537 nm and at liquid helium temperatures gave EPR signals at g = 1.877, 1.951 and 2.045. The protein has an isoelectric point of 3.30, and one (2Fe-2S)-cluster per molecule with a midpoint potential, at pH 7.5, of -410 mV (n=1) . The m olecule of C. reinhardii ferredoxin consists of 95-99 amino acid residues which includes the full com plement of amino acids, being alanine the most abundant.

Fluorescence detected ODMR measurements in zero field on the triplet states of light harvest­ing (LH) pigments in chromatophores from Rhodopseudomonas sphaeroides R 26.1 and Rhodo­pseudom onas capsulata A1a + are reported. In both cases at least two distinct triplet states in the antenna system with zfs values of D between 209 and 217 and E between 56 and 67 x 10 -4 cm -1 are observed.

Derivatives of 6-chloro-5-methylthiopyrimidines provide potent inhibitors of the photosynthe­tic electron flow, which act like Diuron on fluorescence induction kinetics and competitively displace it from its binding site. Structure-activity relationships show that, unlike triazines, ac­tivities of 2-or 4-alkylamino derivatives are restricted by steric hindrances. Decreases in inhibit­ory activities of these compounds observed in triazine-resistant chloroplasts are lower than de­creases reported for triazines themselves.

Herbicidal halogen substituted 4-hydroxypyridines are inhibitors of photosynthetic electron flow in isolated thylakoid membranes by interfering with the acceptor side of photosystem II. Tetrabromo-4-hydroxypyridine, the most active compound found, has a pI 50 -value of 7.6 in the inhibition of oxygen evolution in both the reduction of an acceptor of photosystem I and an acceptor of photosystem II. The new inhibitors displace both metribuzin and ioxynil from the membrane. The 4-hydroxypyridines, like ioxynil, have unimpaired inhibitor potency in Tristreated chloroplasts, whereas the DCMU-type family of herbicides does not. It is suggested that 4-hydroxypyridines are complementary to phenol-type inhibitors, and a common essential element is proposed. The 4-hydroxypyridines do not inhibit photosystem I or non-cyclic electron flow through the cytochrome b/f complex. But they do have a second inhibition site in photosynthetic electron transport since they inhibit ferredoxin-catalyzed cyclic electron flow, indicating an antimycin-like property. A comparison of the in vitro potency of the compounds with the in vivo potency shows no correlation. A major herbicidal mode of action of the group is related to the inhibition of carotenoid synthesis, and access to the chloroplast lamellae in vivo for inhibition of electron transport may be restricted.

The light-induced proton uptake of rod outer segment disc membranes has been investigated in the absence and presence of G-protein. Proton uptake was measured as the alkalisation of the suspending medium using a pH electrode and/or the indicator dye bromocresol purple. It was found that besides the known proton uptake of photolysed rhodopsin additional uptake of one proton accompanies formation of the complex between rhodopsin and G-protein. No measurable proton uptake was found under conditions of rapid redissociation of the complex indicating an only transient protonation during its lifetime. Proton uptake was the same in washed membra­nes recombined with G-protein and in ordinarily stacked rod outer segments. The additional proton uptake reported here is not due to enhanced metarhodopsin II.

Two different cyanobacteria, Anabaena CH 1 and CH2, were isolated from Taiwan paddy soils. Both strains can grow well with daily dilution method. Anabaena CH1 shows a blue-green color and Anabaena CH2 a green brownish one. Nitrogenase activity decreased as cultures were transferred from light to dark. When a darkened culture was placed again into the light, nitrogenase activity recovered within two hours, but not in the presence of chloramphenicol. Energy supply for nitrogenase within both strains was different. Nitrogenase activity of Anabaena CH1 was light-dependent and oxygen in heterocyst was exhausted through oxyhydrogen reaction. Except photosynthesis, respiration may be used as energy source for nitrogenase in Anabaena CH2. Respiration was the major one to protect nitrogenase against oxygen.

Oxidation of methionine, 4-(methylthio)-2-oxobutyric acid (KMB), or 1-aminocyclopropane carbonic acid (ACC) are indicator reactions for activated oxygen species such as singlet oxygen ( 1 O 2 ), OH · -radical like oxidants, superoxide anion (O 2 ·- ), hydrogen peroxide (H 2 O 2 ) or activated hemo-iron complexes like peroxidase-or catalase-“compound”. Methionine is oxidized by OH · as well as by 1 0 2 forming ethylene, but not by tetrachloro-decaoxygen complex (TCDO) in the absence or presence of catalytic hemoproteins such as peroxidase, hemoglobin or myoglobin. Both KMB and ACC are oxidized by TCDO under the catalysis of the above hemo-proteins where neither catalase nor superoxide dismutase are inhibitors. TCDO hemo-protein complex is an oxidant with similar properties as peroxidase-compound I and can clearly be differentiated from 0 2 ·- , H 2 O 2 , OH · and 1 O 2 .

[ 14 C]penicillin binding experiments and membrane analysis were carried out with cell envelope preparations from Escherichia coli and Proteus mirabilis. After incubation with [ 14 C] penicillin G labeled free lipoprotein could be identified. The analysis of the isolated lipoprotein by SDS polyacrylamide gel electrophoresis indicates that there is only one protein with an apparent molecular weight of 7000. The amino acid composition of isolated labeled free lipoprotein from E. coli was identical to the lipoprotein already found in E. coli. It is a point of interest that the amino acid composition of the isolated labeled free lipoprotein from P. mirabilis D52 differs from that found in other mutants of this strain. The free form of lipoprotein from P. mirabilis D52 is composed of 61 amino acids and has glycine, phenylalanine and proline as specific components.

Two-dimensional gel electrophoresis according to O’Farrell [6] was used to study synthesis of membrane bound proteins during outgrowth of Bacillus subtilis spores. Three major classes of membrane bound proteins were distinguished, depending on the time of onset and duration of their synthesis: Ia - a great amount of proteins typical of vegetative cells made throughout all stages of outgrowth and vegetative growth; Ib - vegetative proteins synthesized during middle and later outgrowth stages as well as during vegetative growth (sequential activation); II - outgrowth-specific proteins synthesized during early outgrowth but not in vegetative cells. A similar classification was made for total proteins [9]. In a temperature-sensitive mutant defective in early outgrowth the sequential activation of vegetative genes (group lb) is especially inhibited under restrictive conditions, whereas the syn­thesis of group Ia-proteins and outgrowth-specific proteins (II) could be detected during early outgrowth stages. We suggest that the expression of outgrowth genes, whose products might be found among the outgrowth-specific proteins, could be involved in the sequential reactivation of vegetative genes during outgrowth. The vegetative proteins of group la (continuous synthesis) are produced throughout growth and differentiation. On the other hand the synthesis of vegetative proteins, group Ib , is switched off during sporulation and switched on during outgrowth. The control mechanisms of the expression of these two groups of vegetative genes during growth and differen­tiation are discussed.

1. The effect of ʟ-norleucine, an isomer of leucine, on protein metabolism in vivo was studied in suckling rats. Rats were injected subcutaneously with various doses of ʟ-norleucine (0.5 and 5.0 μmol/g body wt.) every 12 h from 3 to 15 days post partum. Protein concentration, amino acid concentrations, and incorporation of [ 3 H]tyrosine into protein were analyzed in liver, muscles of thigh and small intestine. Amino acid concentrations and insulin levels in serum were also measured. 2. At 5 days of age, norleucine induced an increase in protein concentration of skeletal muscle with an increased incorporation of [ 3 H]tyrosine into protein indicating an accelerated protein synthesis. Changes in protein metabolism were paralleled by alterations in the amino acid pattern of this tissue. 3. When protein concentration and protein synthesis were increased in skeletal muscle, protein concentration of small intestine was decreased, accompanied by elevated levels of amino acids in tissue. Protein synthesis of small intestine was not altered by the norleucine treatment. The results suggest a close interrelationship between skeletal muscle and small intestine with respect to protein turnover. 4. The effects of norleucine were less pronounced at 10 and 15 days of age, which indicates a metabolic adaptation to the treatment. 5. Alterations in amino acid concentrations of tissue due to changes in protein metabolism were not uniform but tissue-specific. 6. Current concepts for explaining the effects of branched-chain amino acids (BCAA) on protein turnover in skeletal muscle are based on the assumption that the BCAA or leucine alone might become rate-limiting for protein synthesis in muscle under catabolic conditions. The amino acid analogue norleucine, however, cannot replace any of the BCAA in protein. Additionally, norleucine affected protein metabolism in highly anabolic organisms. Therefore, the present thoughts on this issue appear to be incomplete.

Locomotor activity patterns of six cavernicolous carabid species representing the course of regressive evolution have been investigated under constant conditions. The analysis establishes new relationships between the circadian structure of the activity patterns observed under constant conditions (LL and D D) and the phylogenetic adaptation to cave conditions. This is due to the mathematical method used for investigating the activity patterns, i. e. the Wigner-Ville spectrum that enables the evaluation of nonstationarities. It overcomes the hypothesis of stationarity which was the base of previous examinations. The new method delivers a more refined gradual decrease of the stability of the circadian pattern of activity in cavernicolous beetles than could be achieved by classical periodogram analysis. It is also shown that the locomotion patterns of extremely evolved troglobitic clearly deviate from a purely signal-free (white noise) structure. Possible time­keeping mechanisms that may cause the measured stabilities and regressive lines of possible evolutionary development are discussed.

In the -γradiolysis of N 2 O/O 2 -saturated solutions of 2′-deoxyguanosine, 2′-deoxyguanosine-5′-aldehyde has been identified as the major product (G = 0.27) which gives rise to a positive 2-thiobarbituric acid test usually associated with malondialdehyde or structurally closely related compounds. From experiments with tetranitromethane as oxidant instead of oxygen it is concluded that the major primary precursor is not the radical at C(5′) of the sugar moiety but a base radical. The relevance of these observations with respect to radiation-induced changes in DNA is discussed.

Prosomes are small cytoplasmic RNP complexes. We present evidence that their RNA is a potential and selective inhibitor of viral mRNA translation while translation of normal cellular mRNA e.g. rabbit globin mRNA or HeLa cell mRNA is not affected.

Xenon as a potential triplet quencher is capable of in­hibiting bluelight-induced, flavin-mediated transport of redox-equivalents across lipid membranes. The experiments described are based on a model system developed recently. Ferricytochrome c as a potential electron acceptor is trap­ped in the lumen of unilamellar lipid vesicles, EDTA* as a potential electron donor added to the bulk volume and amphiphilic flavin as a mediator bound to the vesicle mem­brane. However, Under isotropic aqueous conditions the bluelight-driven electron transport chain comprising EDTA, flavin and Cyt c is not significantly affected by xenon. These data call for the reinterpretation of results obtained with xenon as potential inhibitor of phototropic curvature of grass coleoptiles. which were taken to suggest the flavin singlet as active species.

In isolated spinach thylakoids the hydrophobic photo­reactive probe 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl-diazirine almost exclusively labels the photosystem I reac­tion center and the light-harvesting chlorophyll a/b protein. In isolated cytochrome b 6 /f-complex, all four components of the complex get labeled, but to a different extent. The amount of labeling in the protein components is correlated to the number of polypeptide segments embedded in the membrane system.

Ectocarpene, (+)-(6S)-(1Z-butenyl)-2,5-cycloheptadiene, is the mating pherom one of the three distantly related seaweeds Ectocarpus siliculosus, Sphacelaria rigidula and Adenocystis utricularis. Analogu es of this pheromone, dif­fering in position and π-electron density of the double bond in the C 4 -side chain, were synthesized and assayed for biological activity. The results suggest, that the fundamen­tal molecular recognition process is conservative in all species, while receptor dynamics and -topologies have been modified by individual developments and expressions dur­ing plant phylogeny.

Butterflies have nine photoreceptors within each ommatidium which have been named from their positional orientation across the rhabdom. Within all ommatidia ex­amined, there are four morphologically discrete receptor types: 1. Two dorsal-ventrally aligned cells - the vertical receptors; 2. two anterior-posteriorly aligned cells - the horizontal receptors; 3. four diagonally aligned cells -the diagonal receptors; 4. one basally occuring bilobed cell - the basal receptor. The nine retinula cell axons pass through the basement membrane and enter an optic car­tridge of the lamina ganglionaris as a single bundle. The organization of these axons within their cartridge has been investigated in the nymphalid-type butterfly Agraulis vanillae. Prior to entry into the optic cartridge, the axonal bun­dle undergoes a 90° positional rotation. Retinula axons become grouped into three regions: a dorsal triad and a vent­ral triad of three axons, one horizontal and two diagonal retinula cells each; a central group composed of two verti­cal retinula cells, the basal cell, and 4 -5 lamina monopolar cells. Retinula axons of the triads appear to be coupled by bulbous cytoplasm ic projections. Two types of coupling ap­pear to occur: 1. Within each triad, the horizontal retinula cell axon is coupled to both diagonal axons. 2. The horizon­tal retinula cell axon of one triad is coupled to the horizon­tal cell of the adjacent triad. Because of the 90° reposition­ing of the ommatidial axon bundle, specific cell types from one ommatidium appear to be linked to retinula cells of ommatidia immediately above or below them. These non­conventional receptor cell interactions are described and a possible function in receptor coupling suggested.