Volume 34, Issue 5-6

Verbenalin and a new iridoid, hastatoside (2) have been isolated from Verbena hastata and V. officinalis. The structure and absolute configuration of hastatoside has been determined by spectroscopical methods, and by correlating 2 chemically with verbenaline and the lactone 6. The lactone 7 has been synthesized from verbenaline. The name brasoside-tetraacetate is proposed for 7, since brasoside has been found in V. brasiliensis as a natural product.

12 species of the genus Verbena and one Citharexylum species have been examined for iridoids. Besides the known compounds verbenaline, hastatoside, griselinoside, lamiide, ipolamiide and brasoside, two new iridoids, pulchelloside I (1) [2] and pulchelloside II (2) [3] have been isolated from Verbena species. The structures and absolute configurations of pulchelloside I and II have been determined by spectroscopical methods and some chemical reactions. Citharexylum solanaceum has been shown to contain lamiide and durantoside I. The distribution of the iridoids is in excel­ lent agreement with the taxonomic treatment of the genus Verbena by Schauer. The taxonomic significance of our data and some data from the literature regarding the classification of the sub­ family Verbenoideae is discussed.

The α-, β-, and γ-amanitin content of 11 samples of Amanita verna Bull., collected during 1975 - 1978 in Germany and Switzerland, has been determined by high performance thin-layer chromatography (HPTLC) of crude methanolic extracts. The toxicity (i. v. LD 50 for mice of defatted, lyophilized, methanolic extracts) of 3 samples has been compared with that of A.phalloides from the same site of collection. The amanitin content of A. verna ranged from 2250 to 4570 mg/kg dry weight; the fungi contained almost as much β- as α-amanitin, whereas the γ-amanitin content amounted to about 12% of the total amanitin. A. verna contained less amanitin (65% on the average) than A. phalloides from the same collection site, but it was not significantly less toxic, since the phallotoxins contributed to the toxicity of either species in our tests.

[4- 14 C] Progesterone, fed to leaves and whole plants of Convallaria majalis L., was incorporated into the main cardenolide glycosides. After application of [4- 14 C] progesterone to the leaves of intact plants via the epidermis labelled desglucocheirotoxin, convallatoxin and convallatoxol were isolated from rhizomes and roots. When the precursor was administered to rhizome cuttings no radioactivity could be detected in the cardiac glycoside fraction. The experiments pointed out that during the stage of flowering biosynthesis of cardenolide glycosides takes place in the leaves of Convallaria majalis rather than in the subterranean parts of the plant. A translocation of these substances from the leaves to rhizomes and roots seems possible.

Using 14 C-labelled dieldrin, the resorption, body distribution, and rate of excretion as well as the metabolism was investigated qualitatively and quantitatively. The experiments with mice, rats, rabbits, Rhesus monkeys, and chimpanzees were performed under identical conditions to obtain reliably comparable results.

An improved enzymatic method for the synthesis of adenylylsulfate (APS) from adenosine 5′-phosphate using APS-reductase from Thiobacillus denitrificans is described. Isolation of millimole quantitities of this sulfur nucleotide is achieved rapidly by means of ion exchange chromatography on a strongly basic ion exchange resin. A facile and reproducible desalting procedure is described.

Acetalation of the cis-diol moiety of adenosine or inosine with aliphatic ketoesters of different chain lengths leads to alkylidene derivatives of the nucleosides, which differ in the number of methylene groups in the hydrocarbon chain. By alkaline hydrolysis of the ester group in 1a - c or 3a - c the corresponding acids 2a - c and 4a - c have been prepared. The configuration of the new chiral centres has been determined as R. Enzymatic deamination of the alkylidene derivatives of adenosine leads to the inosine compounds. The rate of deamination reaction is raised by an increasing number of methylene groups in the alkylidene residues or by use of the esters instead of the acids. The alkylidene derivatives of adenosine were coupled with 6-aminohexylagarose yielding polymers with adenosine as ligands. No enzymatic deamination of the polymer with the shortest spacer was observed. The polymers with the longer spacers were converted to the corresponding inosine derivatives. The velocity of the deamination reaction was raised by an increasing spacer length.

The syn-anti equilibrium about the glycosidic bond in adenosine and some related analogues was studied by means of 1 H NMR spectroscopy, with the aid of several model analogues fixed in given conformations either by intramolecular bonding, or by introduction of a bulky substituent. A model unambiguously and exclusively in the syn conformation is 8-(α-hydroxyisopropyl) adenosine; while one fixed in the a n ti conformation is 8,5′-anhydro-8-oxoadenosine. A new analogue, fixed in the high an ti conformation, is 8,2′-O-isopropylidenearabinofuranosyladenine. Several additional new model compounds were synthesized and their properties are described. With the aid of these models, the syn-anti dynamic equilibrium was examined for adenosine and some related compounds in different solvent systems, and the conformer populations evaluated quantitatively. The validity of the procedure applied, and the accuracy of the results, are critically examined, and compared with findings obtained by other procedures. Available literature data on the syn-anti equilibrium in other 8-substituted adenosines are re analyzed in the light of the present results. An analysis is also presented of the interdependence of the various conformational parameters, i. e. conformation about the glycosidic bond and those of the sugar ring and exocyclic carbinol group, in adenosine and 2′,3′-O-isopropylideneadenosine.

Two proteins (P 1 and P 2 , with mol weights of 57,500 and 27,500, respectively) were isolated from Euglena gracilis. Both proteins show cyanide-insensitive superoxide dismutase activity in the “classical” superoxide dismutase assay, using xanthine-xanthine oxidase as O 2 · − generator. If O 2 · − is generated chemically (autoxidation of reduced anthraquinone), photochemically (illuminated riboflavine) or pulse radiolytically, only protein P 1 but not P 2 shows SOD activity. Protein P 1 contains 1 g atom (determined: 0.82) iron (no Mn or Cu) per mole protein and may thus be defined as iron-superoxide dismutase. Protein P 2 , showing the spectral properties of a flavoprotein, exhibits the activities of ferredoxin-NADP-oxidoreductase and “diaphorase”. The cyanide-insensitive SOD-activity of this “diaphorase” in the xanthine oxidase-assay for superoxide dismutase makes this classical and commonly used test unreliable for assaying cyanide insensitive SOD activities. The existence of the “prokaryote-type” of superoxide dismutase (Fe-SOD) in Euglena gracilis is exceptional for an eukaryotic, autotrophically grown organisms.

Yeast Aminopeptidase I, Molecular Forms, Immunological Behaviour Yeast aminopeptidase I, when purified from autolysates of brewer’s yeast, is obtained in two molecular forms a) the enzymatically active dodecameric complex (M r = 640 000, s 20, w = 22 S) and b) inactive hexamers (M r = 320 000, s 20, w = 12 S). Although the amino acid composition of the 12 S protein is very similar to that of the active enzyme, the hexamers behave differently in ionic exchange chromatography and during electrophoresis on polyacrylamide gels. Moreover, the antigenic properties of 12 S and 22 S aminopeptidase forms suggest a considerable degree of structural diversity. Several strains of Saccharomyces cerevisiae did not contain hexameric forms although their 22 S aminopeptidase was immunologically indistinguishable from brewer’s yeast amino­ peptidase. It is proposed that the hexameric protein is the result of “unproductive ” aggregation of aminopeptidase subunits.

Two S-adenosyl-ʟ-methionine:furanocoumarin O-methyltransferases of R . graveolens, acting at the 5-and 8-hydroxyls of the psoralen nucleus, were completely resolved by adsorption on a general affinity ligand, 5 -(3-carboxypropanamido) xanthotoxin, followed by specific desorption by bergaptol and xanthotoxol, respectively. The 5-O-methyltransferase was purified 450-fold by this procedure, the 8-O-methyltransferase 112-fold, and both enzyme fractions were electrophoretically homogeneous. No resolution could be achieved of the activity against two 5-hydroxypsoralens or of the activity against two 8-hydroxypsoralens, and conclusive evidence is presented for the existence of only one 5-O-methyltransferase and only one 8-O-methyltransferase acting on linear furanocoumarins.

For a better insight of the molecular basis of the photobiological effects of furocoumarins, the relevance of proteins oxydation by singlet oxygen produced by these substrates under irradiation with long u. v. light was studied. Complex oligomeric as well as simple monomeric purified enzymes with high or low molecular weight and different properties and simple amino acids were irradiated under oxygen in presence of 8-methoxy-psoralen. The effects on both proteins and amino acids were compared with those obtained under similar conditions with typical photosensitizers as methylene blue and Rose Bengal. The results indicated that the photooxydation of proteins, although possible, appears to play a minor role, if any, in the mechanism of action of furocoumarin.

Citrinin, a mycotoxin, was studied for its effect on artificial membranes (liposomes). Of the three (net positive, negative and neutral) liposomal preparations tested, the membrane carrying a net positive charge was preferentially acted upon by citrinin and in this case even the lowest con­ centration was found to be sufficient for causing a high degree of damage. This is the first report of evidence that citrinin has a direct effect on membranes. It was shown to exhibit concentration dependency in its lytic activity on artificial membranes leading to leaching of trapped 45 Ca 2+ ions.

Diaphragms of mice were traumatized by division parallel or transversally to the muscle fibres. The extent of traumatization was varied by the degree of subdivision. Oxygen uptake was measured by the Warburg technique. Undivided diaphragms as control displayed constant oxygen uptake for the four hours of observation. With divided diaphragms oxygen uptake was unstable. Division parallel to the muscle fibres resulted in continuous decrease of oxygen uptake after a normal initial phase; the decline was significantly steeper with increasing extent of traumatization. Division transversally to the muscle fibres resulted in an initial increase of oxygen uptake followed in a second phase by a continuous decrease. The initial increase as well as the final decrease of oxygen uptake was more pronounced with higher extent of subdivision.

Absorption and fluorescence experiments on pheophytin and chlorophyll containing lipid bilayer vesicles are reported. Pheophytin aggregates on the vesicles are established from an additional red shifted band (at 695 nm) in the absorption spectrum. These aggregates contain pheophytin in an arrangement with the molecular planes of the porphyrin rings being parallel and cover about 10% of the vesicle surface. The lipid phase dissolves pheophytin up to a molar ratio of 15% above the lipid phase transition. This solubility limit decreases hardly on solidification of the lipid. For chlorophyll a containing vesicles the aggregates are not observed in the absorption spectrum. The chlorophyll solubility is about equal to that of pheophytin. This suggests that the phase separa­ tion indicated from fluorescence measurements at temperatures below the lipid phase transition does not lead to the formation of strongly bound chlorophyll aggregates.

A fast oxygen uptake, induced by a sequence of short (5 µsec) saturating flashes was observed in chloroplasts of wild type tobacco and two chlorophyll-deficient tobacco mutants. One of the chlorophyll mutants is the earlier described variegated tobacco NC 95. Chloroplasts of this mutant exhibit only photosystem I mediated photoreactions, hence the observed oxygen uptake is to be associated with photosystem I. This is further substantiated by the fact that the oxygen uptake is insensitive to DCMU in the two chloroplast types used, which have both photosystems fully functioning. The uptake depends on the addition of electron acceptors like p-benzoquinone in intact chloroplasts or on p-benzoquinone or ferricyanide in chloroplasts that have lost the envelope. In dark adapted chloroplasts, therefore, under these conditions the overall apparent gas exchange in the first two flashes is consumption. Although the uptake is slower than photosynthetic oxygen evolution it clearly affects the oxygen yield in the flash sequences. This is demonstrated by several experiments in which the apparent oxygen consumption in the absence of DCMU oscillates with a periodicity of four. We have indications that in chloroplasts of the tobacco aurea mutant Su/su the oxygen uptake is faster than in wild type chloroplasts.

The inhibition of photosynthetic electron flow in broken chloroplasts by dibromothymoquinone and dibromothymohydroquinone (DBMIBH 2 ) is reversed by dithiothreitol (DTT) as well as by serum albumin. The reversal of DBMIBH 2 inhibition by DTT shows a time lag, that of DBMIB only, when chloroplasts and DBMIB had been preilluminated. This is to show that chloroplasts reduce DBMIB and that probably DBMIBH 2 is the actual inhibitor species. Bromonitrothymol, ioxynil and related inhibitory phenolic compounds have a different relation­ ship of inhibitory potency to chemical structure than DCMU and the analogous triazinone herbicide metribuzin but nevertheless inhibit photosynthetic electron flow at the same functional site. This is supported by the finding that labelled metribuzin is displaced from the thylakoid membrane by bromonitrothymol and ioxynil indicating identical binding sites. On the other hand inhibition by the phenolic inhibitors bromonitrothymol and ioxynil but not that of DCMU and metribuzin has a time lag of about 4 min.

An antiserum to violaxanthin in hibits photosynthetic electron transport between water, iodide or tetramethylbenzidine and various electron acceptors in chloroplasts from green tobacco (Nicotian a tabacum var. John William’s Broadleaf). However, electron transport from manganese or diphenyl-carbazide to these acceptors is not impaired. The typical photosystem I reaction from DPIP / ascorbate to anthraquinone-2-sulfonate in the presence of DCMU shows no inhibition. From this it is concluded that the effect of violaxanthin on the photosynthetic electron transport chain lies on the oxygen-evolving side of photosystem II before the site from which diphenylcarbazide or manganese donate electrons. In the presence of DCMU after preillumination we find an effect of the antiserum on fluorescence. The reaction of the antibodies to violaxanthin with stroma-freed chloroplasts depends on the condition of the thylakoid membrane. Chloroplasts which are still swellable react in a bivalent manner and are agglutinated. Non swellable chloroplasts react only in a monovalent manner. This specific binding was demonstrated by means of the Coombs-test. From these reactions it follows that the antigenic determinants of violaxanthin are accessible to the antibodies, hence, they must be located in the outer surface of the thylakoid membrane.

An in vitro transcription system using nuclei from freely suspended callus cells of Petroselinum crispum is described. The use of a filtration technique allowed the measurement of transcription and release of RNA simultaneously over very short time intervals. The transcription showed a biphasic time course, with an early maximum at 2 and a later one at 20 minutes. The early maximum was ascribed to the activity of polymerase II (α-amanitin sensitive), the later to that of polymerase I. While the transcriptional process was independent of the temperature used for incubation up to 26 °C and even increased with temperatures above that, the release of RNA transcribed was inhibited by temperatures above 36 °C.

The sequence complexity of nuclear RNA from mouse liver, mouse spleen and highly malignant P815 mastocytoma was measured by nRNA driven hybridization to unique DNA sequences of P815 cells. The unique DNA sequences represent 63% of the total nuclear DNA of P815 cells and their availibility in hybridization experiments was found to be 76%. Of these sequences 7.8% formed hybrids with nuclear RNA of this cell, about 11.5% with mouse spleen and about 14.5% with mouse liver nuclear RNA. Assuming an asymmetrical transcription, the complexities of these transcripts are 2.8 × 10 8 nucleotides for mouse P815 mastocytoma, 4.3 × 10 8 for mouse spleen and about 5.3 × 10 8 nucleotides for mouse liver. Cellular specifity of the transcribed information was analyzed in additivity experiments, in which unique DNA sequences, not complementary to the nuclear RNA of one cell were annealed to the nuclear RNAs of the two other tissues/cells. In these experiments most of the nuclear RNA se­ quences of P815 cells were found to be also present in the nucleus of mouse liver and spleen. Only a small portion of the unique DNA sequences of P815 mastocytoma (about 1.2% corresponding to 4.4 ×10 7 nucleotides) was found to be complementary only to P815 mastocytoma nuclear RNA.

Chromatin was isolated from rat liver cells at 0, 3, 5, 11, 18 and 24 h following partial hepatectomy. Consistent with findings in cultured cells stimulated to proliferate, there was an increase in chromatin molar ellipticity measured at 276 nm, and a decrease in thermal stability 3 to 8 h after surgery. These events occured prior to the onset of DNA synthesis. These early changes be­ tween non-proliferating (G 0 ) and proliferating (G 1 ) cells, as well as later chromatin conformational changes observed at S and G2 phases, mimic changes in template activity. Results with sheared and unsheared chromatin (both with in vitro and in vivo systems) prove that structural and functional changes can be caused by even the slightest shearing during chromatin preparation, suggesting the loss of native chromatin organization. To eliminate this problem, experiments were also conducted using chromatin in situ. A flow cytometer (FCM) was used to study unfixed liver cell suspensions stained with ethidium bromide (EB). Fluorescence was mea­ sured in the green spectral range after addition of increasing amounts of EB. Experimental evidence is provided that the same alteration in chromatin conformation can be best detected using low molar ratios of EB per unit DNA due to greater fluorescence emission in G 1 respect to G 0 cells. These correlated studies demonstrate that the same changes controlling chromatin organization in situ are detected also in the tertiary-quaternary structure of “isolated” chromatin. These changes in chromatin conformation are macromolecular events related to cell proliferation both at the G 0 -G 1 and G 1 -S transitions.

5-Bromo-2-deoxyuridine, DNA, Chloroplasts, Fern 5-Bromo-2-deoxyuridine (BUdR) is incorporated into chloroplast DNA of Anemia phyllitidis. A possible connection between previously published effects of BUdR and on chloroplast ultrastructure and transitions caused by the base analogue in the DNA is referred to.

Using T-lymphocyte (T-LC) and granulocyte colony (GC) assays with truly proliferating cells, the dose-response relationships of spermine and spermidine, of fetal calf (FCS), calf (CS), horse (HS) and human AB serum (A B S), and of the polyamines in the presence of selected does of the sera were analyzed. In contrast to earlier observations using [ 3 H] thymidine uptake measure­ ments it was found that the polyamines as well as the sera themselves inhibit T-LC and GC growth in the presence of autologous HS and ABS, respectively. The polyamines also inhibit in the presence of additional HS and ABS, yet most effectively with added FCS and CS. Thus the previously reported species-specificity of the serum-dependent polyamine inhibition has here been shown to be more quantitative than qualitative. These studies stress the significance of assays utilizing truly dividing cells and broad dose-response relationships in order to assess specific biological effects in vitro.

A newly synthesized phospholipid, 1,2-O-Dipentadecylmethylidene-glycerol-3-phosphoryl-(N-ethyl-amino) -ethanolamine, having positive charge was found to exercise fusion activity on plant proto­ plasts. This phospholipid is not found in cells and has an unusal chemical structure with a dioxolane ring. Some properties of this compound are discussed in relation to phospholipid-induced cell fusion.

The optics of the posterior eyes of the amphibious spider, Dolomedes aquaticus, are described. The lenses have strongly-curved spherical front and rear surfaces, and therefore, a fairly high light capturing ability - they have F-numbers of 0.9 - yet, the large amount of spherical aberration predicted by their shape is mostly corrected. The receptive segments of the receptors are arranged in rows, each row separated by pigmented glial cells, but within a row, rhabdomeres from neigh­ bouring cells are contiguous so that coupling between cells from the same row seems a possible way of increasing absolute sensitivity for night vision. When the spider submerges, an air-layer is held over the comeal surfaces - as with a diver’s face mask - so that light is focused on to the receptive layer of the retina under water as well as above. These features are considered as adaptations for the nocturnal and semi-aquatic habits of the spider.

The body temperature, activity-rest time, electrolytes of urine samples and mood was measured in two persons during a 19 day period under continuous light conditions in the arctic (vicinity of Ny Ålesund, Svalbard-Spitsbergen). For temperature recording a new thermoprobe and a portable printer was used. Possible week Zeitgeber of the 24 hour day did not synchronize the circadian system, since circadian rhythms of about 26 hours were found. These results open up the pos­ sibility to study effects of drugs on the circadian system of humans under Svalbard conditions.

Temperature dependence of biological processes is described by the modified Arrhenius equation, in which the constant is replaced by a temperature-variable coefficient. The theoretical foun­dation for this replacement is given and experimentally verified.

An algorithm for phylogenetic trees’ construction is analyzed.

A sensitive fluorometric test for the determination of monooxygenase activity in liver microsomes from rats is described. The assay is based on the O -demethylation of 6,7-dimethoxycoumarin (scoparone) to 7-hydroxy-6-methoxy-coumarin and 6-hydroxy-7-methoxycoumarin. Pretreatment of rats with phenobarbital or polycyclic hydrocarbons (benzo[a]pyrene, 3-methylcholanthrene) causes a significant increase in the amount of microsomal dealkylation activity.

β, β-Carotene-2-one was isolated in minute amounts from Cerura vinula and identified on the basis of spectra, hydride reduction, and chromatography. The implications on the biosynthesis of 2-hydroxylated carotenoids in in sects are discussed.

N,N-Dialkyl Ammonium Salts of Saturated Heterocyclic Amines, Antimicrobial Activity Antimicrobial activity of N-alkyl-N-dodecylpiperidinium bromides and N-ethyl-N-dodecylheterocycloalkyl ammonium bromides (pyrrolidine, morpholine, perhydroazepine) deter­ mined on grampositive and gramnegative bacteria, yeasts and moulds, presented as minimum inhibition concentration (MIC). Comparison of the effect of change of structure: lenghtening of alkyl chain, change of heterocyclic ring. Change in the lenght of alkyl chain markedly affects the antimicrobial activity, change of heterocyclic ring has no substantial effect. The most active compounds were N-heptyl-and N-hexyl-N-dodecylpiperidinium bromides.

Incorporation of Na 2 35 SO 4 by ovine brain Ficoll-prepared synaptosomes were studied as a function of time, temperature and substrate concentration. The rate of uptake obeyed Michaelis-Menten kinetics and apparent K m value of 4.4 mᴍ Na 2 SO 4 was determined. In the presence of p-hydroxymer-curibenzoate a reduction in sulphate uptake was noted which suggests that thiol groups may possibly be implicated in the uptake mechanism.

The potential photoaffinity reagents 2,5-diazido-3-methyl-6-isopropyl-1,4-benzoquinone (DAZMIB) and 2,5-diazido-3,6-dimethyl-1,4-benzoquinone (DAZDMQ), which are analogs of the well-known inhibitor 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB), block photosynthetic electron transport in spinach chloroplasts. On irradiation, at relatively high concentrations, DAZMIB inhibits electron flow irrevers­ibly. However, when [ 3 H] DAZDMQ was photolyzed with chloroplasts, no labeling of the membrane proteins was ob­ served. Rearrangements of the nitrenes derived from the oxidized or reduced azidoquinones that might account for this behaviour are discussed.