Volume 42, Issue 5

Alkane distribution patterns were determined in the epicuticular wax of 7 species of Ericaceae, including 5 species from more than one location. Results showed good agreement between sam­ples of any given species from different locations; thus any differences in environmental condi­tions including altitude were insufficient to affect the alkane patterns. Average chain length was highest in Calluna vulgaris and lowest in Vaccinium uliginosum.

The production of essential oil in Chrysanthemum balsamita L. reveals a seasonal dependence. – We conclude that, in blossoms, a dominant influence due to the ontogenetic development of head flowers regulates the production of essential oil. – Freshly harvested head blossoms produced 5 times the amount of oil when compared to fully developed head flowers. Dried leaves contain 5 to 7 times the amount of oil compared to fresh leaves. The composition of the oil from leaves changes more distinctly during the season than the oil from head flowers. Main components of the oil from flowers and leaves are carvon and thujon. During spring and fall, the percentage of sesquiterpenoid hydrocarbons is increased mainly in the oil from leaves. The component β-cubeben can no longer be detected in dried leaves.

Laurencia obtusa, Red Algae, Rhodomelaceae, Acetylenic Ether Epoxy-trans-isodihydrorhodophytin, a new halogenated acetylenic bicyclic ether, has been isolated from a Laurencia obtusa variety, collected in the Marmara sea. Its structure was determined by spectroscopic methods and X-ray diffraction analysis.

An isoflavonoid obtained from dried roots of the Thai legume Pueraria mirifica has been identified by chemical and spectroscopic methods as 7,2′,4′-trihydroxy-5′-methoxy-6′-(3,3-dimethylallyl)isoflavone (kwakhurin).

A C-glucosyltransferase from Fagopyrum esculentum seedlings catalyzes the transfer of glucose from UDP-glucose or ADP-glucose to 2-hydroxyflavanones. In cell-free enzyme preparations it was shown that only 2-hydroxyflavanones, e.g. 2,4′,5,7-tetrahydroxyflavanone and 2,5,7-trihydroxyflavanone were appropriate substrates. Naringenin, naringenin-chalcone and the flavones apigenin and chrysin cannot act as glucosyl acceptors in C-glucosyl-flavonoid biosynthesis. This demonstrates that C-glucosylation occurs after oxidation of flavanones.

The effect of pH changes on the excretion of ammonia and glycolate from algal cells into the medium was investigated in L-MSO (final concentration, 0.5 mм) -treated High-and Low CO 2 -cells of Chlorella vulgaris 211-11 h. The excretion was analyzed in the condition in which the cells were continuously gassed with air at 25 °C. At the values tested, generally more ammonia was excreted in L-MSO-treated Low CO 2 -cells than in L-MSO treated High CO 2 -cells. In both kinds of algal cells more ammonia was excreted at low pH-values and absolutely no ammonia was excreted at pH 8. In the dark, no or only slight ammonia excretion was observed in both L-MSO-treated High and Low CO 2 -cells. Under all these conditions no or only very low glycolate excretion was observed in both L-MSO treated High and Low CO 2 -cells. In High CO 2 -cells rates of photosynthesis were high at pH 6 and lower at higher pH values. On the other hand Low CO 2 -cells showed practically little dependence of photosynthetic rates on the pH. This result might indicate that the major part of the ammonia excretion observed, was not due to the inhibition of photosynthesis at acid pH values. It is known that ammonia excretion in L-MSO treated algal cells is due to the inhibition of the refixation of ammonia which originates from the glycine-serine aminotransferase reaction in the glycolate pathway. Our results demonstrate that glycolate production and glycolate metabolism are more intense at low pH values when compared to high pH values. This is valid for both High and Low CO 2 -cells. Low CO 2 -cells in Chlorella vulgaris 211-11 h exhibit a more active glycolate metabolism than High CO 2 -cells.

Arylsulfatase activity was detected in the green alga Chlorella fusca 211-8b during sulfur starvation. No activity was present during growth on sulfate, whereas activity was found with sulfur sources as growth-limiting factor including L-cysteine and L-methionine. Arylsulfatase activity was found after transfer to sulfate starvation conditions after a lag-period of about 3 h; addition of cycloheximide prevented arylsulfatase formation, suggesting the participation of protein synthe­ sis. Addition of sulfate to cultures with active arylsulfatase led to a decay of this enzyme, whereas addition of sulfate and cycloheximide prevented this decay suggesting that protein synthesis is also needed for degradation of arylsulfatase activity. The K m for p-nitrophenyl sulfate was determined to be 0.8 mм. The activity of the arylsulfatase is not affected by sulfate or phosphate, however it is inhibited strongly by sulfite, cyanide and boric acid and therefore is classified according to Dogson and Spencer (Meth. Biochem. Anal. 4, 211-254 (1957) as an arylsulfatase of type I.

In root hairs from seedlings of Glycine max cultivars, isolated from the root system and com­ pared with the complete organ, specific soluble proteins have been found. By FPLC chromatography and SDS gel electrophoresis root hair specific proteins with molecular weights of 13, 21, 34, 38 and 42 kDa were separated. Additionally, proteins with molecular weights of 12, 20, 69 and 74 kDa were significantly enriched in root hairs compared to roots without root hairs. By using CNBr activated Sepharose with antibodies against the root system without root hairs, the pres­ ence of root hair specific proteins was confirmed in extracts from isolated root hair cells. Enrichment of Fe and Ca in some of the proteins from the root hairs is demonstrated. The present knowledge of the biochemical specificity of legume root hairs, the target cells of Rhizobium and Bradyrhizobium infection, is discussed.

One of the carboxyl groups of o-succinylbenzoic acid (OSB, 1) is activated during the biosyn­ thesis of vitamin K2 and anthraquinones. In an attempt to determine the exact structure of the naturally occurring OSB coenzyme A thioester, the three possible esters were prepared via the imidazolides of OSB. HPLC systems were developed for the successful separation of the esters and their structural analogues. The structures of the coenzyme A esters were investigated and their products of hydrolysis determined quantitatively. Two of the esters were highly labile at pH 7 and 30 °C. UV spectroscopy is a suitable method to distinguish between different positions of activation. Fast atom bombardment mass spectrometry was successfully employed to charac­ terize two of the esters. The data strongly indicate that the “aliphatic” coenzyme A ester is the intermediate in vitamin K 2 and anthraquinone biosynthesis.

In soil-grown 3-to 4-weeks-old Phaseolus coccineus L. plants the chloride content changed antagonistically in the extensor and flexor of the laminar pulvinus during the circadian leaf movement in continuous light. This is as expected for an osmoticum involved in the volume changes of pulvinar cells. H ow ever, the malate content of extensor and flexor cells was not altered in a circadian manner. Furthermore, during light/dark cycles the malate content in both, extensor and flexor, was higher in the light than in the dark. This indicates that malate was not used in the osmotic motor of leaf movement and that its level was not controlled by the circadian clock in 3-to 4-weeks-old soil-grown plants. When leaves were cut from 14-days-old soil-grown plants and cultured in distilled water the pulvini were depleted of chloride and the malate content was increased. In these chloride de­ prived leaves malate, and to a lesser extent citrate (about 1/10 of malate), changed antagonistically in a circadian manner in the extensor and flexor, indicating that these organic anions were now involved in the osmotic motor and under the control of the circadian clock. The similar properties of pulvinar and stomatal movements of starch-containing guard-cells are evident: in both cases, depending on the availability of chloride, CL - and/or organic anions are used for the compensation of the electrical charge of K + .

Corynespora cassiicola DSM 62474 and Diplodia gossypina ATCC 10936 were found to cleave some acyclic terminal terpendiols and prolongate them later by addition of a C 2 -unit to afford 1,2-dihydroxy-propyl-compounds. The enzymes involved in this reaction from both microorganisms displayed a strong stereoselectivity which is completely different in both strains. The stereochemi­cal requirements of the substrates for both strains and the mechanism were elucidated by using closely related substrates. The absolute configuration of the diols were solved by correlation with com pounds of known absolute configuration.

Sn-glycerol-3-phosphate Sn-glycerol-3-phosphate accumulates during starch catabolism by intact isolated chloroplasts from Chlamydomonas reinhardii in the dark. Further products detected mainly were 3-phospho-glyceric acid and triose phosphates with a ratio between 3-phosphoglyceric acid, sn-glycerol-3-phosphate and triose phosphates of 1.0:0.9:0.2. The production of sn-glycerol-3-phosphate by a plastidic glycerol-3-phosphate dehydrogenase provides a reasonable mechanism for regenerating reducing equivalents during continuous starch breakdown in the chloroplast of the alga C. reinhardii.

The carbon flow from 3-phosphoglycerate to pyruvate and acetyl-CoA within the chloroplast as well as the pathway for the formation of β-carotene, plastoquinone-9 etc. as plastidic isoprenoids from photosynthetically fixed CO 2 hitherto remained unclear because the presence of the phos- phoglycerate mutase in chloroplasts had not unequivocally been proven. To clarify this question, the incorporation of 14 CO 2 , [2- 14 C]acetate, [2- 14 C]pyruvate, [2- 14 C]glycine, [3- 14 C]serine and [2- 14 C]mevalonate into β-carotene, plastoquinone-9, sterols and fatty acids was studied using spinach and barley protoplasts and barley seedlings. In protoplasts as well as in seedlings under conditions of CO 2 fixation, the largest portion of the acetyl-CoA derived compounds formed was β-carotene and plastoquinone-9 rather than fatty acids and sterols. High rates of fatty acid synthesis were obtained by supplying acetate as well. Mevalonate was incorporated into sterols but not into β-carotene and plastoquinone-9. Direct evidence was obtained for the hitherto questioned plastidic phosphoglycerate mutase. Low activities were found in spinach chloroplasts which were substantiated by the criteria of latency method. Substrate flow from 3-phosphoglycerate to pyruvate via 2-phosphoglycerate and phosphoenolpyruvate was shown by applying [1- 14 C]- glycerate and following its incorporation. From this the following conclusions were drawn: (i) The plastidic IPP synthesizing system is strongly separated from the cytosolic-ER one. (ii) As the formation of β-carotene and plastoquinone-9 is favoured under conditions of photosynthetic CO 2 fixation, a direct carbon flow from 3-phosphoglycerate to isoprenoids via acetyl-CoA by a low- capacity pathway, which shows high affinities for the substrates, is suggested.

Chlorophyll a fluorescence quenching was studied in intact, CO 2 fixing chloroplasts isolated from spinach. Energy-dependent quenching (q ᴇ ), which is correlated with the light-induced pro­ ton gradient across the thylakoid membrane presumably reflects an increase in the rate-constant of thermal dissipation of excitation energy in the photosynthetic pigment system . The extent of q ᴇ was found to be linearly related to the decrease of quantum yield of photosynthesis. We suggest that this relationship indicates a dynamic property of the membrane to adjust thermal dissipation of absorbed light energy to the energy requirement of photosynthesis.

UV absorption spectroscopy revealed the presence of MDA with characteristic maximum at about 266 nm in the dialysate of the UV irradiated liposomal suspension. The amount of MDA released increased linearly with increasing UV dose. The fluorescence method for the assay of MDA was found nearly 1500-fold more sensitive than the TBA colour reaction method.

Conversion of uridine and cytidine to their 5′-O-tosyl derivatives, followed by cyanation with tetraethylammonium cyanide, reduction and deamination, led to isolation of the hitherto un- known homouridine (1-(5′-deoxy-β-D-allofuranosyl)uracil) and homocytidine (1-(5′-deoxy-β-ᴅ-allofuranosyl)cytosine), analogues of uridine and cytidine in which the exocyclic 5′-CH 2 OH chain is extended by one carbon to CH 2 CH 2 OH. Homocytidine was also phosphorylated to its 6′-phosphate and 6′-pyrophosphate analogues. In addition, it was converted, via its 2,2′-anhydro derivative, to arahomocytidine, an analogue of the chemotherapeutically active araC. The structures of all the foregoing were established by various criteria, including 1 H and 13 C NMR spectroscopy, both of which were also applied to analyses of the solution conformations of the various compounds, particularly as regards the conformations of the exocyclic chains. The behaviour of the homo analogues was examined in several enzymatic systems. Homo- cytidine was a feeble substrate, without inhibitory properties, of E. coli cytidine deaminase. Homocytidine was an excellent substrate for wheat shoot nucleoside phosphotransferase; while homouridine was a good substrate for E. coli uridine phosphorylase. Although homoCMP was neither a substrate, nor an inhibitor, of snake venom 5′-nucleotidase, homoCDP was a potent inhibitor of this enzyme (K i ~6 μм). HomoCDP was not a substrate for M. luteus polynucleotide phosphorylase. None of the compounds exhibited significant activity vs herpes simplex virus type 1, or cytotoxic activity in several mammalian cell lines.

Oligonucleotide-streptolysin S complex inactivated by alkali treatment remains nonhemolytic, even after acidification and mixing with intact carrier oligonucleotide rich in guanyl residue. Upon dehydration, how ever, the inactive streptolysin S-oligonucleotide mixture turned to be hemolytic, and this reactivation of the hem olysin was promoted by treatment with guanidine hydrochloride. After alkaline hydrolysis, streptolysin S was freed from nucleotide moiety, by gel filtration through a Sephadex G -50 column. From this nonhemolytic apotoxin as well, active streptolysin S complex was reconstructed upon dehydration with the carrier.

1-β-ᴅ-Ribofuranosylpyrrolidin-2,5-dione (9) was synthesized by ribosylation of N-silylated succinimide (7) with 1,2,3,5-tetra-O-acetyl-β-ᴅ-ribofuranose in acetonitril in the presence of tin tetrachloride. The compounds 9, 1-β-ᴅ-ribofuranosyl-l-H-pyrrol-2,5-dione (5) and N-methyl- maleinimide (2) were converted with ammonia to the ring-opened components 16. 14 and 15. The bioreactivity of the N-maleinimide derivatives 2 and 5 with respect to addition and ring-opening reactions with amino acid side chains containing either thiol or amino groups was shown in model reactions with glutathion (compds. 17,18) and lysine (compds. 19, 20). The ring opening reaction of 3-methyl-3-phenyl-1-β-ᴅ-ribofuranosylpyrrolidin-2,5-dione (11) with lysine yields 21, thus demonstrating the possibility of glycosuccinylation of amino groups in proteins.

Synthesis and spectroscopic analysis of some alkylated DNA purine bases are described. HPLC separation methods are developed for the determination of DNA alkylation rates in mammalian embryonic tissues. Following treatment of pregnant mice with the ethylating agent ethyl-methanesulfonate (EMS), an appreciable amount of alkylation (ethylation and methylation) was found in the nuclear DNA of the embryos during organogenesis. The results are discussed in context of our thesis that a certain amount of DNA alkylation in the embryos is correlated to the teratogenic potential of alkylating agents.

The sting of the Neoguinean pseudomyrmecine ant, Tetraponera sp. (group punctulata) is modified in a structure resembling a drawing pen. These ants do not sting, but smear upon enemies a contact poison. The venom which originates from the poison gland is a mixture of eight alkaloids with an original tricyclic ring system. The structure of the major component, tetra-ponerine-8, has been determined by X-ray diffraction analysis. The poison of Tetraponera is highly efficient in deterring aggressive sympatric ants and tetraponerine-8 shows toxic properties against Myrmica rubra when applied topically, with a LD 50 of 5 × 10 -4 mg/mg of ants.

By means of electroantennography and single cell recordings, GC and GCMS analyses and GC analysis with EAG detection (6E,11 Z)-6,11-hexadecadienal, (6E,11 Z)-6,11-hexadecadienyl acetate and (4E,9Z)-4,9-tetradecadienyl acetate were identified as the primary components of the sex pheromone of female Antheraea pernyi (Lepidoptera: Saturniidae).

Levels of 24 free amino acids were estimated in the brain after administration of 5,6-dihydroxy-tryptamine and 6-hydroxydopamine into the lateral brain ventricles of male Wistar rats. These neurotransmitters caused serotoninectomy and sympathectomy in the diencephalon, striatum, brain stem and medulla, thalamus and hypothalamus, cerebral cortex and cerebellum. The most abundant amino acids in these brain structures were: glutamic acid, serine, aspartic acid, cystine, gamma-aminobutyric acid, glycine, tryptophan and alanine. We detected and quantified changes in the levels of these and other amino acids in the investigated regions of the rat central nervous system, under the influence of these two neurotransmitters.

The effect of hydrostatic pressure on calcium dependent p-nitrophenyl phosphate hydrolysis of the sarcoplasmic reticulum calcium transport enzyme has been investigated at different degree of enzyme saturation by calcium and Mg-p-nitrophenyl phosphate to distinguish between activation and binding volumes. The enzyme saturated by both ligands displays a significant dependence of the activation volume on pressure, rising from 20 ml/mol at atmospheric pressure (0.1 MPa) to 80 ml/mol at 100 MPa. At subsaturating concentration of Mg-p-nitrophenyl phosphate an activation volume of 35 ml/mol prevails between 0.1 and 40 MPa. At subsaturating concentration of calcium the activation volume approximates 80 ml/mol in the same pressure range. The binding volume for both substrates is likewise pressure dependent falling from 20 ml/mol to 0 ml/mol for Mg-p-nitrophenyl phosphate and rising from 67 ml/mol to 155 ml/mol for calcium. The pressure dependence of activation and binding volumes is analysed on account of a simplified reaction scheme yielding activation volumes and rate constants for individual reaction steps.

Nitrate reductase (EC 1.6.6.2) has been isolated from 11 Sphagnum (Bryophyta) species and purified up to 500-fold by a procedure involving (NH 4 ) 2 SO 4 precipitation and blue sepharose affinity chromatography. The purest fraction showed a nitrate reductase activity of 7.7 nkat mg protein -1 with NADPH as the electron donor. Beside NADPH and NADH, FMNH 2 and - after blue sepharose affinity purification of the enzyme - MVH 2 were used as electron donors in different assays.

3,4-Dihydro-9-hydroxy-3-methylisocoumarin [R(-)-mellein] was identified as the major component of the male wing gland pheromone of the bumble-bee wax moth, Aphomia sociella L. (Pyralidae, Galleriinae). The presence of Aspergillus ochraceus Wilhelm, a mellein producing microorganism, was detected in the intestines of last-instar larvae and in bumble-bee nests. Arguments for a new type of insect-fungus relationship are presented.