Band 48, Heft 11-12

The 1 H and 13 CNMR spectra of some representative biflavonoids have been reinvestigated by proton-detected C-H correlation. Some assignments made in earlier publications are revised. Rules for the influence of one flavonoid moiety upon the chemical shift of the other’s protons are given.

10-Hydroxymajoroside, a second representative of the rare majoroside type iridoid gluco-sides, along with the well known aucubin, has been isolated from Plantago cornuti.

The design and synthesis of a series of methyl 5(6)-substituted benzimidazole-2-carbamates as possible anthelmintics have been described. A rational analysis of the structural parameters which segregate the activity of resulting benzimidazole-2-carbamates against enteric and tissue dwelling helminths is presented. The influence of single and multiple spacers, which link the pharmacophores at 5(6)-position of benzimidazole-2-carbamate, on the activity against Ancylostoma ceylanicum (hookworm), Syphacia obvelata (pinworm), Hymenolepis nana (tapeworm), Litomosoides carinii and Acanthocheilonema viteae (filarial worm) has been presented. This analysis indicates that for activity against intestinal helminth the presence of one spacer holding the pharmacophore approximately 3 Å apart from the parent nucleus is usually preferred. While for activity against tissue dwelling parasite, the repetition of the benzimidazole-2-carbamate nucleus joined together through the 5,5′-position with one spacer kept apart by distance of 3 Å unit is usually desired.

Three new aseptic cultures of Marchantia polymorpha from different collecting sites have been induced from the gametophytes. The formation of bisbibenzyls in differentiated cultures and cell suspension cultures was investigated. We observed that the production of the main bisbibenzyl, Marchantin A was induced by lack of nitrate and the addition of cupric sulfate to the medium. The comparison of bisbibenzyl patterns in three different cultures suggest the existence of chemical races of M. polymorpha. Bisbibenzyl patterns in differentiated cultures and cell suspension cultures appeared to be similar.

The isolation, structural elucidation and biological activities of oligosporon (1), oligosporol A(2) and oligosporol B(3), three new antibiotics from cultures of the predacious deuteromycete, Arthrobotrys oligospora, are reported. The structures were elucidated by means of high resolution mass and high field NMR spectroscopy. The compounds exhibited weak antimicrobial, cytotoxic and hemolytic effects, but were not active towards the nematode Caenorhabditis elegans. Other Arthrobotrys species were also found to produce these or similar compounds.

Ethanolic extracts of Propolis are used as antiinflammatory and wound healing drugs since ancient times. In order to facilitate a comparison of different extracts, the standardization on the basis of quantitative determination of prominent components of these extracts has been substituted for simple biochemical “activity” tests. One of these activity tests bases on the in­ hibition of peroxidase-catalyzed oxidation of indole acetic acid indicating the presence of a defined mixture of monophenolic and diphenolic compounds. Other tests (diaphorase-catalyzed reductions and xanthine oxidase-catalyzed oxidations) demonstrate significant radical scavenging properties. Water-soluble extracts of propolis exhibit higher antioxidative and inhibitory activities as compared to the ethanolic extract.

Ethanolic and aqueous extracts of the “bee glue” Propolis exhibit antioxidative properties and are used as antiinflammatory drugs in folk medicine. In order to standardize the principle activities of prominent components of these extracts, simple biochemical tests have been introduced in the preceding paper. These activity tests prove the high antioxidative and inhibitory capacities of aqueous and ethanolic extracts of propolis in vitro. In the present communication we report on experiments documenting photodynamic quenching properties of these extracts. Using riboflavin, rose bengal or hematoporphyrin as photoactivators and ketomethyl-thiobutyric acid or crocin as indicators, the protective functions of propolis preparations can be demonstrated. The results indicate that the aqueous extracts are more active than the corresponding ethanolic preparation.

Cocaine is abundant in leaves of Erythroxylum coca var. coca Lam. Consequently, cocaine and two of its early biosynthetic precursors were monitored for 24 h to determine whether they fluctuated diurnally. E. coca was grown under controlled environmental conditions in a growth chamber in soil at pH 3.5. After 17.5 months of growth, leaves not less than 35 days old were harvested every 2 and 4 h for 24 h for arginine, phenylalanine and cocaine content, respectively. The content of cocaine was determined by GC/MS and amino acid content by HPLC. Diurnal fluctuation of cocaine occurred during the 24 h cycle. Cocaine was highest in leaves of E. coca at 8 and 16 h where its content was 7.67 and 9.45 mg·g -1 dry weight, respectively. Arginine and phenylalanine in leaves of E. coca also displayed diurnal rhythmic patterns of fluctuation. The content of arginine declined from hours 6 to 12 and increased to the highest content at 13 h (21.8 mg·g -1 dry weight). Overall, phenylalanine content was lower than arginine, but had two peak periods of accumulation during the 24 h cycle, occurring at 8 and 14 h. The content of phenylalanine in leaves of E. coca during the peak hours was 6.98 and 6.54 mg·g -1 dry weight, respectively.

Glutathione in its oxidized form (GSSG) is rapidly taken up into isolated barley vacuoles, while uptake of reduced glutathione (GSH) is only marginal. GSSG transport is strictly ATP-dependent and is a saturable process (K m 0.4 to 0.6 mм). Uptake is inhibited by vanadate, but not by bafilomycin, a specific inhibitor of the vacuolar H + -ATPase. The observation that i) the non-hydrolysable ATP analogue adenylylim idodiphosphate does not stimulate, but rather inhibits the ATP-dependent uptake of GSSG and ii) that a four-fold accumulation of GSSG is observed in the presence of bafilomycin, suggests that GSSG is transported into the vacuole by a primary active process similar to that of glutathione S-conjugates (E. Martinoia, E. Grill, R. Tommasini, K. Kreuz, N. Amrhein, Nature 364, 247-249 (1993)). U ptake of GSSG is competitively inhibited by the glutathione S-conjugate of the herbicide m etolachlor (K i 80 μм), indicating that transport of GSSG is mediated by the glutathione S-conjugate ATPase.

Cu(II)-Induced oxidation of catechols (dopa, dopamine and epinephrine) to yield indolic aminochromes is speeded up by at least two orders of magnitude upon addition of DNA. This effect was observed both in aerobic and anaerobic solutions, indicating that it is not due to the reductive activation of oxygen with formation of O 2 ·- and H 2 O 2 as oxidant. It is suggested that the unfavourable redox equilibrium of the catechol-Cu(II) complex, ROH-Cu(II) ⇄ Cu(I) + RO · + H + , to yield Cu(I) and semiquinone, is efficiently driven to the right by DNA due to formation of the strong DNA-Cu(I) complex, for which a stability constant of K ≈ 2×10 9 м -1 has been estimated previously. Absorbance changes characteristic to the formation of DNA-Cu(I) appear in fact concomitantly with the 480 nm absorption of aminochrome, derived from the semiquinone RO · . Cu(II)-Induced oxidation of ascorbate and o-phenylenediamine (leading to 2,3-diaminophenazine) was also speeded up by almost two orders of magnitude in presence of DNA. The observed activation of redox reactions of copper in the vicinity of DNA may have important implications with respect to the toxicological properties of certain bioreductive endogenous and xenobiotic compounds.

n-Propyl N-(3,4-dichlorophenyl)carbamate, which induces flowering while it inhibits a step or steps in the phenylpropanoid metabolism in Asparagus officinalis L. seedlings, was found to retard the conversion of t-cinnamic acid to p-coumaric acid by high-pressure liquid chromatography of the metabolites in the shikimic acid pathway. The concentrations of the metabolites preceding t-cinnamic acid on the pathway in treated and untreated seedlings were the same, but those of p-coumaric acid and later metabolites were significantly lower in treated plants. The carbamate inhibited phenylpropanoid metabolism when used to treat stem segments that included a shoot apex primordium, where flowers are induced, and when added to a 100,000 × g microsomal fraction prepared from such segments. NADPH-cytochrome P-450 and NADH-cytochrome b 5 reductases in the 100,000 × g fraction were not inhibited by the carbamate. The results showed that this compound has its site of action on the endoplasmic reticulum and that it inhibits cytochrome P-450s, including t-cinnamic acid 4-hydroxylase. We examined the flower-inducing activity of known cytochrome P-450 inhibitors, and found that piperonyl butoxide also causes flowering.

Formation of major prenylquinones and carotenoids was investigated by comparing the incorporation of [ 14 C]mevalonate into segments of different age from green and etiolated leaves of 22 C-grown rye seedlings (Secale cereale L.) and from 32 C-grown rye leaves which contained bleached and proplastid-like ribosome-deficient plastids, due to a heat-sensitivity of 70S ribosome formation. The contents of plastidic isoprenoids were much lower (between 2 - 30%) in the achlorophyllous than in green leaves. In green leaves [ 14 C]mevalonate incorporation into non-polar lipids and into plastoquinone was partially inhibited in the presence of gabaculin, an inhibitor of chlorophyll synthesis. However, except for β-carotene, [ 14 C]mevalonate incorporation into isoprenoids continuously increased with age also in achlorophyllous etiolated or 32 °C-grown, as in green, leaves and was, except for P-carotene and plastoquinone, higher in etiolated than in green leaves. In bleached °32 C-grown leaves [ 14 C]mevalonate incorporation into all plastidic isoprenoids was strikingly (up to 45-fold) higher than in green control leaves. While degradation of P-carotene was greatly enhanced in bleached 32 °C-grown leaves, relative to green control leaves, and could thus compensate for a higher apparent synthesis, chase experiments did not reveal any marked differences of the turnover of other isoprenoids. The half times of plastoquinone. phylloquinone and lutein were in the order of 2-3 days. Within a 24 h chase period a-tocopherol degradation did not become apparent. Uptake of [ 14 C]mevalonate and [ 14 C]isopentenyl pyrophosphate by isolated bleached plastids from 32 °C-grown leaves was much more rapid than by chloroplasts and resulted in higher precursor accumulation within the organelle. While mevalonate incorporation into isoprenoid lipids was not detected, isopentenyl pyrophosphate was incorporated into isoprenoid lipids, including plastoquinone. Rates of incorporation by isolated chloroplasts or bleached plastids were of similar order. The results illustrate that divergent types of plastid differentiation are associated with fundamental developmental changes of the metabolic flow of isoprenoid precursors between different products and compartments and, in particular, with changes of import into the plastid compartment.

The effect of diethyl pyrocarbonate (DEPC) on the photosynthetic electron transport was investigated in isolated spinach thylakoids by partial electron transport rate and thermolumi- nescence measurements. Incubation of thylakoids at pH 6.5 with 5 mм DEPC for 15 min resulted in a considerable inhibition of electron transport from water to dichlorophenolindo- phenol. The inhibition was only partially releaved by addition of the donor, diphenylcarbazide indicating the effect of DEPC both on the donor and acceptor sides of PS II. In the thermoluminescence glow curve DEPC-treatment abolished the B band (S 2 Q B - radiative charge recombination) at 30 °C with a concomitant appearance of the Q band (S 2 Q A - charge recombination) at 10 °C. This suggests that in isolated thylakoids possessing an active water-splitting system DEPC affects the electron transfer from Q A to Q B but does not inhibit the electron transport from manganese to Q A during the S,→S 2 transition of the water-splitting system. At the acceptor side of PS II the targets of DEPC are probably the histidines which are coordinated to the non-heme iron. Illumination of thylakoids at -80 °C following DEPC addition after two preflashes at 5 °C resulted in the replacement of the A(A T ) thermoluminescence band at -30 °C with a band appearing at -15 °C. This observation can be explained by the effect of DEPC on a donor side histidine component participating in the generation of the A(A T ) band. Consequently, in the interpretation of results obtained by DEPC treatment of PS II, both the donor and acceptor side effects of DEPC should be considered.

Apoplastic, cytoplasmic, ionically and covalently bound isoperoxidases were investigated from needles of Norway spruce (Picea abies (L.) Karst.) and a pronounced genetic variability of the isoenzyme pattern was found. The enzymatic activity of each group of isoperoxidases was determined between November 1989 to November 1990 from needles of three locations (Velmerstot, Glindfeld, Haltern) with different ozone concentrations. Apoplastic peroxidases of needles from trees grown in Velmerstot and Glindfeld showed up to two-fold higher activities than the same group of peroxidases from Haltern. Based on the recorded increased ozone concentrations in Velmerstot and Glindfeld a positive correlation between the activity of the apoplastic peroxidases and ambient ozone concentrations at the different locations could be demonstrated. A putative role of the apoplastic peroxidases as a bioindicator for air pollutants is discussed.

During a five-year period, components of the thylakoid membrane in needles of the second generation of undamaged and damaged trees of Norway spruce were studied at three different mountain sites in West Germany. Visible signs of damage at these sites are a yellowing of the light-exposed sides of the needles as well as the loss of needles. The goal of this study was to determine damage-induced alterations in composition and physiological reactions of the thylakoid membranes in spruce needles. In order to meet this purpose, contents of chlorophyll a and b, electron transport rate of photosystem II, contents of the D 1 protein, cytochrome f, as well as P-700 were measured. The chlorophyll content in the needles of the damaged spruce trees was significantly lower than in the needles of the undamaged trees. In addition to this, the typical annual course of chlorophyll content was exclusively observed in the needles of the undamaged spruce trees. If related to dry weight, a drastic reduction of the electron transport rate and of the redox components of the thylakoid membrane was observed due to damage, indicating a degeneration of the photosynthetic membranes. The contents of D1 protein and the photosynthetic electron transport rates were also markedly reduced in the needles of the damaged trees, when related to chlorophyll content of thylakoids, suggesting an early and particular impairment of photosystem II. The comparison of spruce trees showing different signs of damage demonstrates that certain biochemical parameters concerning the photosynthetic membranes (chlorophyll, cytochrome f, ratio photosystem II/I) reflect the extent of damage and are suitable for an early indication of a beginning, but still invisible damage of spruce trees.

Three V A mycorrhizal fungal species were isolated from soils in Alberta, Canada and examined by scanning electron microscopy and energy-dispersive X-ray microanalysis. Mature spores of Glomus aggregatum developed an outer hyaline wall which contained lower levels of calcium than the middle wall. Examination of G. pansihalos spores revealed a lower level of calcium in the outer evanescent wall as compared to the ornamented wall. When spores of Entrophospora infrequens were examined, the wall of the vesicle was found to contain similar levels of calcium as the ornamented wall of the spore. The significance of the results concerning the presence of calcium in mycorrhizal spore walls is discussed, as is the occurrence of the mycorrhizal species.

Asocainol (ASOC) belongs to class I of the antiarrhytmic drugs, i.e., those that exert their action at the level of the sodium channels of the myocardial cell membrane. It has been suggested that their molecular mechanism of action might be through nonspecific interactions with phospholipids that surround the channel proteins. In order to test this hypothesis, ASOC was made to interact with two multibilayer systems, one built-up of dimyristoylphosphatidylcholine (DMPC) and the other of dimyristoylphosphatidylethanolamine (DMPE). These are the type of lipids that are respectively found in the outer and inner monolayers of human erythrocytes. The experiments were carried out in a hydrophobic as well as in a hydrophilic medium below the phospholipid main transition temperatures. The perturbing effect of ASOC upon the bilayer structures was determined by X-ray diffraction. It was found that ASOC was able to fluidize DMPC in both media but not to DMPE.

Analysis of the marine sponge Verongia aerophoba from the Canary Islands afforded eight brominated secondary metabolites including the small molecular weight compounds aeroplysinin-1 (5) and the dienone (7) which were previously shown to arise by enzymatically catalyzed degradation of aerophobin-2 (4) and isofistularin-3 (1) following breakdown of the cellular compartmentation of the sponge. All compounds were identified from their NMR and mass spectra. Aeroplysinin-1 as well as dienone which arise from isofistularin-3 or aeropho­ bin-2 by biotransformation within the sponge showed a significantly higher antibiotic as well as cytotoxic activity than their biogenetic precursors. Antibiotic activity was studied with re­spect to several gram-positive and gram-negative bacteria including B. subtilis, S. aureus and E. coli. The MICs (MBCs) of aeroplysinin-1 (5) and the dienone (7) varied between 12.5-25 (50-100) μg/ml respectively. Cytotoxicity was tested in vitro towards HeLa cells, a human cervix uteri tumour cell line. Aeroplysinin-1 (5) and the dienone (7) displayed pronounced cytotoxic activity with IC 50 s of 3.0 and 3.2 μм respectively. A five-fold increase in cytotoxicity was observed after O-acetylation of the dienone (7). The IC50 of the dienone O-acetate (0.6 μм) was comparable to that of the clinically used anticancer drug cisplatin (0.7 μм).

Interactions of novobiocin (NB) and nalidixic acid (NA) with thiols were investigated in vitro in thymic (T-) and splenic (S-) cells of the rat, by determining nucleic acid synthesis as well as nucleoid sedimentation and viscosity of alkaline cell lysates. In T-cells NB, at concentrations of 0.35-1.4 mм, increased unscheduled D N A synthesis (UDS) and RNA synthesis (RNS), whereas S-cells underwent a dose-dependent inhibition of UDS and RNS following exposure to NB at concentrations > 0.7 mм. Combining NA and thiols (e.g., dithiothreitol) resulted in a slight stimulation of UDS in S-cells and in a highly significant increase of UDS in T-cells ascribed to a depletion of the cellular thymidine pool. In both cell types, neither NB nor NA exerted a significant effect on thiol-induced DNA damage. At a concentration of 1.4 mм, NB increased the viscosity of alkaline T-cell lysates; the opposite effect was observed in S-cells. From these results as well as from previous investigations we conclude that T-and S-cells differ in their state of chromatin conformation. This interpretation offers a simple model for the study of the influence of chromatin structure on cell-specific physico-and/or chemico-biologi-cal interactions.

From Dicranum scoparium 5′,3‴-dihydroxyamentoflavone and 5′,3‴-dihydroxyrobustaflavone were isolated. The compounds were identified spectroscopically.

The sticky lipophilic exudates produced by glands on inflorescences of Rosa centifolia cv. muscosa and Rubus phoenicolasius contain flavonoid aglycones. Ten different flavonoids were identified from Rosa centifolia cv. muscosa, including several flavones with 6,8 -methoxy substitution. In Rubus phoenicolasius, the major flavonoids are methyl ethers of quercetin.