Volume 50, Issue 1-2

In a search for new inhibitors of RNA-directed DNA-polymerases kuehneromycin A (1) was isolated from fermentations of a Tasmanian Kuehneromyces species. Its structure was elucidated by spectroscopic methods. Kuehneromycin A (1) is a non-competitive inhibitor of avian myeloblastosis virus (K i 200 μᴍ) and moloney murine leukemia virus (K i 40 μᴍ) reverse transcriptases. The second compound, kuehneromycin B (2) is a strong inhibitor of platelet aggregation stimulated with different inducers. In addition, both compounds exhibit cytotoxic and antimicrobial activities.

The long-chain alkyl esters homologues of the cuticular needle wax of Picea abies consist of a series of isomers, which are formed by different n-fatty acids and n-alkanols of various chain lengths. Even-numbered esters are dominating. Methyl esters range from C 27 to C 37 , even-numbered esters are only minor components. Furthermore, a new class of homologue wax esters was isolated. Examination by gas chromatography, mass spectroscopy and chemi­cal studies resulted in identification of pentenyl esters of triacontanoic, dotriacontanoic, tetratriacontanoic and hexatriacontanoic acid.

Brassica juncea, Brassica carinata and Sinapis arvensis resistant lines to Leptosphaeria maculans and four Brassica napus cuitivars susceptible to this pathogen in seedling stage were analyzed in relation to the accumulation of phytoalexins after inoculation with L. maculans. Cotyledon inoculations with spore suspensions of an aggressive and a non-aggressive isolate of L. maculans were performed on seedlings of these lines. The quantity of accumulated phytoalexins in the cotyledons was determined at different time intervals after inoculation. The content and composition of phytoalexins differed considerably among the tested species and according to the fungal isolate used. In the tested Brassica species seedling resistance against L. maculans could not be related to phytoalexin accumulation. However, in Sinapis arvensis phytoalexins might contribute to the resistance reaction to this pathogen.

Kinetic mechanism of purified spinach leaf NAD(P)H glyoxylate reductase (GR-1) was studied using either NADPH and NADH as alternative substrates with glyoxylate. The mech­anism was elucidated from substrate kinetic patterns using NADH as a cofactor rather than NADPH. With NADPH varied versus glyoxylate, and with NADPH and glyoxylate varied at a constant ratio, the patterns obtained on double reciprocal plots appeared to be consistent with a ping-pong mechanism; however, kinetic patterns with NADH conclusively ruled out the ping-pong reaction in favour of the sequential addition of the reactants. Product inhi­bition studies with glycolate and NADP have suggested either that NADPH binds to the enzyme before glyoxylate or that the addition of substrates is a random one. Studies with active group modifiers suggested an involvement of histidine, serine and cysteine residues in GR-1 activity. Salts had little or no effect on the activity of the enzyme, with the exception of cyanide, which had an apparent K i of ca. 2 mᴍ. Studies with several metabolites used as possible effectors of GR-1 activity have suggested that the enzyme is modulated only by substrate availability in vivo. The apparent insensitivity of GR-1 to metabolic effectors is consistent with the proposed role of the enzyme in detoxifying glyoxylate which may act as a potent inhibitor of photosynthetic processes in plant tissues.

In cucumber cotyledons (Cucumis sativus L.) containing several soluble and particulate forms of lipoxygenase (LOX), the location of LOX forms in microsomes has been studied. We concentrated on the question whether the plasma membrane contains one or more forms of LOX. As methodology, we applied both the two-phase partition with polyethylene glycol/dextran and density gradient flotation of plasma membrane-enriched membrane fractions. Both methods show that a high percentage of the microsomal LOX can be attributed to the plasma membrane. Emphasis was put on the findings that the LOX located at the plasma membrane consisted of a species behaving as an integral membrane protein and another form characterized as a peripheral membrane protein by solubilization with carbonate. With long distance SDS-PAGE and immunodecoration using anti-lipid body LOX antiserum, it is possible to distinguish between microsomal LOX forms by small but significant differences in size. Treatment of seedlings with methyl jasmonate led to an enhanced level of LOX at the plasma membrane.

Arginine residues of soybean lipoxygenase-1 was modified with an arginine-directed chemical modifier, 2,3-butanedione. Although inactivation was not visible if the enzyme reaction was monitored under the standard assay condition (83.3 μᴍ linoleic acid dispersed in 200 mᴍ sodium borate, pH 9.0), rapid inactivation was observed with 5 mᴍ sodium borate, pH 8.0. The inactivation was protected by the addition of a substrate, linoleic acid, in the modification mixture. Kinetic analyses indicated that one arginine residue accounted for the inactivation. Enzymological analyses showed that the modification narrowed the pH-activity profile of L-1 and made L-1 sensitive to salt concentration of the assay solution. Strong inactivation by modification was found at low salt concentration and low pH. This was not due to a physical change of the linoleic acid. On the other hand, product specificity of L-1 was not altered after modification. Taken together, the modified arginine residue(s) was thought to be not essential to the catalysis but have an important role in supporting an ideal electrostatic interaction within L-1 and/or between L-1 and a substrate even in sub-optimal reaction conditions.

Microsomal preparations from Rauwolfia serpentina Benth. cell suspension cultures cata­lyze a key step in the biosynthesis of ajmaline -the enzymatic hydroxylation of the indole alkaloid vinorine at the allylic C-21 resulting in vomilenine. Vomilenine is an important branch-point intermediate, leading not only to ajmaline but also to several side reactions of the biosynthetic pathway to ajmaline. The investigation of the taxonomical distribution of the enzyme indicated that vinorine hydroxylase is exclusively present in ajmaline-producing plant cells. The novel enzyme is strictly dependent on NADPH 2 and O 2 and can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole and carbon mon­oxide (the effect of CO is reversible with light of 450 nm). This suggests that vinorine hy­droxylase is a cytochrome P450-dependent monooxygenase. A pH optimum of 8.3 and a temperature optimum of 40 °C were found. The Km value was 3 μᴍ for NADPH 2 and 26 μᴍ for vinorine. The optimum enzyme activity could be determined at day 4 after inoculation of the cell cultures in AP I medium. Vinorine hydroxylase could be stored with 20% sucrose at -28 °C without significant loss of activity.

Anthranilate synthase and chorismate mutase activities which control the flow of substrate from chorismate into the tryptophan pathway and into the phenylalanine/tyrosine pathway, respectively, were examined in three near isogenic wheat lines of Triticum aestivum L. (cv. Prelude Sr 5, highly resistant to stem rust infection; cv. Prelude Sr 24, moderately resistant; cv. Prelude srx, susceptible). The activities of both enzymes were found to increase in re­sponse to inoculation with the stem rust fungus Puccinia graminis f. sp. tritici or treatment with Pgt elicitor. Thus, both the tryptophan branch and the phenylalanine branch appear to contribute to the resistance response in wheat leaves. Only the cytosolic but not the plastidic fraction of the enzyme activities appears to be affected by fungal infection or elicitor treat­ment. Some differences with respect to degree and time dependency of enzyme activation were noticed between the three wheat lines following inoculation but not after treatment with the Pgt elicitor.

Light-driven electron transport in liposome-bound photosystem II (PS-II) particles be­tween water and ferricyanide was monitored by bare platinum electrode oxymetry. The modi­fication of the experimental system with the exogenous quinones α-tocopherol quinone ( α-TQ) or plastoquinone (PQ) resulted in a pronounced effect on photosynthetic oxygen evolution. The presence of α-tocopherolquinone ( α-TQ) in PS-II samples decreased the rate of red light-induced oxygen evolution but increased the rate of green light-induced oxygen evolution. Blue light applied to the assay system in which oxygen evolution was saturated by red light resulted in a further increase of the oxygen signal. These findings are interpreted in terms of a cyclic electron transport around PS-II, regulated by an excitation state of β-carotene in the reaction centre of PS-II. A mechanism is postulated according to which energetic coupling of β-carotene in the reaction centre of PS-II and that of other antenna carotenoid pigments is regulated by the portion of the xanthophyll violaxanthin, which is under control of the xanthophyll cycle.

Leaves from 145-year-old and 44-year-old beech trees were harvested during 1991-1993. Chlorophyll (Chl) and redox components of the thylakoid membrane, including P-700, cytochrome f (Cyt f) and D 1 protein, were determined with the following results. Chl a + b, P-700 and Cyt f per unit of fresh weight (FW), dry weight (DW) and leaf area (LA) increase significantly during leaf development. This can be attributed to a massive membrane syn­thesis and new thylakoid formation in the cells. The Chl a/Chl b ratio decreases with the synthesis of Chl during the development of beech leaves and is reduced further with Chl breakdown in the stage of senescence. When expressed on the basis of Chl, a reduction in the amount of P-700 and Cyt f is observed during the growth stage, indicating a marked enlargement of the functional photosynthetic unit. During senescence of beech leaves, Chl breaks down, and P-700 and Cyt f per unit of FW, DW and LA decrease dramatically with the decline in Chl levels. However, when expressed on the basis of Chl, P-700 remains rela­tively constant, while Cyt f shows a pronounced increase. The uncommon pattern of Cyt f change in senescent beech leaves and the relationship between the amount of Chi and the content of Cyt f throughout an entire growth period of beech leaves are discussed.

Thylakoids isolated from SAN 9785 (4-chloro-5-dimethylamino-2-phenyl-3(2H)-pyridazi-none)-treated pea plants showed an inhibition of “state transition” and the light-harvesting complex II (LHC II) phosphorylation-mediated changes in the energy distribution between photosystem II (PS II) and photosystem I (PS I) as measured by a decrease in PS II and an increase in PS I fluorescence yield. Interestingly, in these thylakoids the extent of phosphorylation-induced migration of light-harvesting complex (LHC II-P) to non-appressed mem­brane regions was only marginally inhibited. We propose that the suppression in the ability for “state transition” by SANDOZ 9785 (SAN 9785) treatment occurs due to a lack of effec­tive coupling of the migrated LHC II-P and PS I. Since we observed a decrease in the antenna size of PS I of the treated plants, the lack of effective coupling is attributed to this decrease in the antenna size of PS I.

We have analysed the thermoluminescence (TL) properties of two lines of Euglena exhibiting two degrees of resistance to diuron, by a factor of 100 (ZR 25) and 1000 (ZR 250) respectively, as compared to wild type line (Z). In addition, the two ZR lines developped an identical resistance to atrazine since the I 50 for this herbicide in each line was 75 times larger than in wild type. Special TL characteristics were evidenced in the two lines. Bands after 2 flashes (or more) showed a shift of the peak maximum towards low temperature, the shift being the largest in the most DCMU-resistant cells. Similar results were obtained with isolated thylakoids, except that the TL bands appeared at a temperature higher than in corresponding cells. Oscillations in the amplitude of the bands in a flash sequence were largely damped in cells (and thylakoids), particularly in the most DCMU-resistant lines. The results are interpreted as indicating accumulation of Qᴀ − Qʙ after flashes due to a decrease of the equilibrium constant for the reaction Qᴀ − Qʙ ⇄ QᴀQʙ − accompanying the DCMU resistance.

A physiologically active substance has been isolated from Brazilian propolis and charac­terized as a new clerodane diterpenoid, as indicated by human hepatocellular carcinoma HuH 13 cell cytotoxicity assays. This compound inhibited growth of the hepatoma cells at a concentration around 10 μg/ml and arrested the tumor cells at S phase as revealed by flow cytometry. At higher concentrations it exerted lethal damage. The compound showed cyto­toxicity on human lung carcinoma HLC-2, HeLa, KB and rat W3Y cells, whereas human diploid foreskin and primary rabbit kidney cells were less affected.

A test system is described, which allows the search for compounds interfering with human sex hormone-binding globulin (SHBG) even in complex plant extracts. The method has been evaluated and applied to Urtica dioica root extracts. The lignan secoisolariciresinol (5) as well as a mixture of isomeric (11 E)-9,10,13-trihydroxy-11-octadecenoic and (10 E)-9 ,12,13-trihydroxy-10-octadecenoic acids (3 and 4, resp.) were demonstrated to reduce binding activity of human SHBG. Methylation of the mixture of 3 and 4 increased its activity about 10-fold.

Ribonucleosides are secreted as products of cellular RNA and ribonucleotide metabolism into physiological fluids such as blood, milk and urine. Unmodified and modified ribonucleo­sides have been detected in the micromolar range as minor constituents in the milk of differ­ent mammals. In addition to the common nucleosides adenosine, cytidine, guanosine, inosine and uridine, modified components such as 1-methyladenosine, 1-methylguanosine, 1-methylinosine, N2-methylguanosine, N2-dimethylguanosine, N6-carbamoyl-ʟ-threonyladenosine, pseudouridine and 5-aminoimidazole-4-carboxamide-N-ribofuranoside (AICAR) have been identified and most of them quantified in samples of human and/or bovine and/or goat’s milk. From these investigations it is known that nucleosides, in analogy to nucleotides, show a typical species-specific pattern. Longitudinal studies have been carried out to determine the concentration profiles of the individual ribonucleosides in the milk of humans as a function of the nursing time.

Sorbitol and mannitol, two stereoisomeric osmolytes, inhibit the ATP-dependent Ca 2+ transport in inside-out vesicles derived from basolateral membranes from kidney proximal tubules. This inhibition (I 0,5 = 400 and 390 mᴍ respectively) cannot be attributed to an in­ crease in Ca 2+ permeability, since the rate of EGTA-stimulated Ca 2+ efflux from preloaded vesicles is not modified by these osmolytes. In the presence of 1 ᴍ sorbitol or mannitol, Ca 2+ uptake is inhibited by 70 and 75%, respectively. Since the Ca 2+ -stimulated ATPase activity is unaffected, sorbitol and mannitol uncouple the Ca 2+ transport from the ATPase activity. The inhibition of Ca 2+ transport by these osmolytes is reversible, since the inhibition dis­appears when the vesicles are preincubated with 1 ᴍ sorbitol or mannitol and then diluted 25-fold in reaction medium to measure Ca 2+ accumulation. On the other hand, these osmo­lytes protect the (Ca 2+ +Mg 2+ )ATPase from the inhibition of Ca 2+ transport and ATPase activity by urea and guanidinium. These data suggest that the high concentrations of polyols that renal cells accumulate during antidiuresis, may regulate Ca 2+ transport across the plasma membrane. In addition, polyols may protect the (Ca 2+ + Mg 2+ ) ATPase from the deleterious structural effects of urea, a compound that also accumulates during antidiuresis.

The uropygial gland waxes of the South American red-legged Seriema (Cariama cristata (L., 1766)) were found to be composed of unbranched alcohols and 2,2′-dialkyl-substituted acetic acids which so far have not been found in skin lipids. When used as a chemosystematic character, the occurrence of this lipid class separates the order Cariamiformes (Seriemas) from all other avian orders hitherto investigated, especially from the Gruiformes (cranes and rails) to which they have been tentatively attributed in the past. From the GC retention time data now available for a series of 2-alkyl-substituted fatty acid methyl esters relative retention time indices for other compounds may be predicted.

Oncopeltus fasciatus (Hemiptera) is morphologically and physiologically adapted to seques­ter cardiac glycosides for its chemical defence against predators. As a prerequisite for carde­nolide utilization this bug has to take up and store the dietary cardenolides. Using isolated midguts it could be shown experimentally that the resorption of [ 3 H]digoxin is time-, and temperature-dependent. The hydrogen ion concentration for uptake is optimal between pH 5.5 and 7 and an activation energy of 45 to 49 kJ/mol can be derived from an Arrhenius plot. Uptake can be inhibited competitively by the polar cardiac glycoside convallatoxin. Sulfhydryl group reagents (N-ethylmaleimide), membrane destabilizing compounds and respira­tory chain inhibitors reduce digoxin resorption, as do inhibitors of sugar transport (e.g., phloridzin). The experimental data thus provide evidence for a cardenolide carrier in midgut cells of O. fasciatus.

The effect of dietary γ-hexachlorocyclohexane (lindane) (50 -350 ppm, 0.17-1.19 μmol/kg chow) on the activity of enzymes of lipogenesis, viz., fatty acid synthase (FAS; EC 2.3.1.85), citrate cleavage enzyme (CCE; EC 4.1.3.8), malic enzyme (ME; EC 1.1.1.40), glucose-6-phosphate dehydrogenase (G 6 PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (PGDH; EC 1.1.1.44), and on serum lipid levels, was investigated in livers of 35-day-old male Wistar rats. Lindane (150 ppm) caused a substantial decline of enzyme activities within the first 24 h of treatment. The decrease was transient, however, and enzyme activities subsequently recov­ered despite continuation of lindane feeding. The recovery of enzyme activities was compara­ tively fast in the case of ME, G 6 PDH and PGDH, but very slow with FAS and CCE. Activities of lipogenic enzymes decrease when animals are starved, and increase much beyond prestarvation levels upon subsequent refeeding. Lindane in the refeeding diet blunted this overshoot of FAS and CCE activities in a dose-dependent manner. In contrast, activities of ME, G 6 PD H and PGDH responded to low dietary lindane concentrations with a substantial stimulation of the increase of activity, whereas at high lindane concentrations the overshoot was inhibited. According to their responses to lindane exposure, liver lipogenic enzymes could be grouped into 2 categories with FAS and CCE representing one and ME, G 6 PDH and PGDH representing the other group. Polychlorinated biphenyls (PCBs) in the diet caused basically opposite changes of the activities of the lipogenic enzymes. Co-administration of lindane and PCBs resulted in an apparent cancellation of effects, suggesting that lindane and PCBs affect fatty acid synthesis at opposite points. Levels of the serum triglycerides were increased significantly as a result of lindane feeding, while serum cholesterol and phospholipid levels were only slightly elevated. The increase of serum triglyceride levels that is routinely observed after refeeding of starved animals was stimulated even more by low concentrations of lindane in the refeeding diet, but inhibited by high concentrations.

The recognition and the activation mechanism of the H 3 histamine receptor was studied based on quantum-chemical calculations. A mechanistic model proposed both for recognition and activation stage clarifies different properties of histamine and α-methylhistamine at the H 3 receptor. Interaction with a hypothetical receptor sites leads to the opening of the intramolecular hydrogen bonding in histamine, whereas the α-methylhistamine remains in closed conformation.

An attempt is made to answer the question posed by the title of this paper. First we show that in primitive self-replicating oligoribotide systems, selection depended from the very start on the existence of 4 kinds of ribotides, forming 2 complementary pairs. Further selection required that the condensation reactions involving the two last positions of the 3′-end of a growing oligoribotide fragment and the first position of the 5′-end of another fragment were catalyzed by randomly synthesized peptides. This established a codon - amino acid concen­tration correlation and clinched triplet segments as the basis of the translation process. Finally, physical arguments are given to show that the monochirality of the ribotides arose from stereochemical reasons, as firstly described by Wald, but that of the amino acids is the result of natural selection acting during the peptide-assisted stage of oligoribotide growth.