Volume 40, Issue 3-4

Monoterpene derivatives with spasmolytic activity inhibited acetylcholinesterase (ACHE, EC 3.1.1.7) in a dose dependent manner in vitro. The median inhibitory concentrations ranges between 10 -4 ᴍ and 10 -2 ᴍ. A significance of our finding may by the possible explanation of effects of monoterpene derivatives seen in distony of digestive tract.

Exogenous ʟ-tyrosine was incorporated 13 -14 times less effectively than exogenous L-phenylalanine into flavonoids in excised cotyledons of 3 days old buckwheat seedlings but proved to be an only 5 times poorer precursor when the experiments were carried out with 5 days old material. Simultaneous administration of ʟ-phenylalanine markedly reduced incorporation of ʟ-tyrosine into flavonoids. A similar decrease in the utilization of exogenous ʟ-tyrosine for flavonoid bio­ synthesis occurred in kinetin-treated cotyledons. However, in cotyledons treated with high doses of glyphosate, an inhibitor of the shikimic acid pathway, an increase in the formation of flavonoids from exogenously supplied ʟ-tyrosine was observed. Under all conditions the relative incorporation rate of exogenous L-tyrosine was highest for anthocyanins and lowest for C-glycosylflavones while within the latter class of compounds the luteolinic derivatives orientin and isoorientin incorporated more label than their apigeninic analogues vitexin and isovitexin. PAL and TAL activities were found to be present in the cotyledons in a ratio of 50:1. The possible role of ʟ-tyrosine as an alternative natural precursor for the biosynthesis of flavonoids and other related polyphenols is discussed.

Chalcone synthase activity was found in enzyme preparations from spinach. In homogenates of young leaves two different activities of the enzyme could be separated by DEAE-ion exchange chromatography and chromatofocusing. Both activities formed naringenin with [2- 14 C] malonyl- CoA and 4-coumaroyl-CoA as substrates. They exhibited only slight differences in substrate specificity. For both activities 4-coumaroyl-CoA proved to be the most suitable substrate at both pH 6.8 and 8.0. Eriodictyol and homoeriodictyol formation from caffeoyl-CoA and feruloyl-CoA, respectively, only occured at pH 6.8. The formation of naringenin by the two activities was maximal at pH 7.5-8.0 and dependent upon the DTE-concentration in the assay mixture.

The amount of streptolysin S produced by resting streptococci was considerably increased after incubation of the washed bacteria with trypsin or pronase. Production of both cell-bound and free forms of the toxin was enhanced by the protease treatment. By addition of trypsin, streptolysin S yield was considerably increased in growing culture as well. Treatment with lysozyme was ineffective, and the toxin production was only slightly promoted by preincubation with hyaluronidase or chymotrypsin. In contrast, pretreatment witn chymotrypsin caused in­creased production of an extracellular nuclease, whereas the yield of this enzyme was reduced after incubation of the cocci with pronase. Evidence was obtained indicating de novo synthesis of the exotoxin in the protease-treated bacteria.

The synthesis of β-galactosidase in Rhizobium meliloti WU60 was found to be inducible by lactose and its non-metabolizable analogue. isopropyl-β-ᴅ-thiogalactoside (IPTG). In contrast to Escherichia coli, galactose and melibiose were very weak inducers of this enzyme in R. meliloti. The maximum level of β-galactosidase in this bacterium is 2% of that in fully induced E. coli. In addition to glucose, the induced synthesis of this enzyme in R. meliloti was repressed by galactose, glycerol, and succinate. In comparison to E. coli, addition of cyclic AMP to the growth medium of R. meliloti did not alleviate the repressive effect of the above compounds on β- galactosidase synthesis. High concentrations of sodium succinate (100 mᴍ) were inhibitory to the growth of R. meliloti. Spontaneous succinate-resistant mutants could be isolated at low frequency. In contrast to the wild type parent, in a succinate-resistant mutant, the synthesis of β-galactosidase was not repressed by succinate.

The oxidation of cysteine with 0 2 is facilitated by isolated membranes of the cyanobacteria Anacystis nidulans and Anabaena variabilis, and further stimulated by Fe 3+ . This reaction accelerates with increasing the pH value and is suppressed by cyanide, benzylhydroxamate, hydroxylamine, but not azide. The agents mentioned inhibited the respiration of the membranes with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) effectively, but did not influence their nonenzymic oxidation. It is inferred that ascorbate in combination with TMPD is oxidized via membrane oxidases. Cysteine oxidation apparently proceeds non-enzymatically and is catalyzed by the cations of heavy metals.

A glyceollin I accumulation of about 6000 pmol · mg dry weight - , a tenfold increase above control root tissue, was found in one type of nodule from Glycine max which had been infected with a fix - strain (61-A-24) of Rhizobium japonicum. In nodules infected with one other ineffective (fix - ) strain of Rhizobium japonicum (RH 31-Marburg) or with two fix + strains of Rhizobium japonicum (61-A-101 and USD A 110) no increase in glyceollin I concentrations above control values was found at either 20 d or 34 d after infection. Nodules infected with Rhizobium japonicum 61-A-24 are distinguished by an early loss of the peribacteroid membrane in the infected host cell, whilst the bacteroids themselves remain stable.

Acetate concentration in spinach (Spinacia oleracea L.) leaf tissue has been determined by direct measurement in a leaf extract to be 70 μᴍ or less. Additional evidence for such low acetate levels came from isotope dilution experiments where the [l- 14 C]acetate incorporation into fatty acids was assayed using purified chloroplasts in the presence of tissue extracts. Pyruvate dehydrogenase complex activity was shown to be present in spinach chloroplasts and was determined by a newly developed acetyl coenzyme A trapping system (100 nmol acetyl CoA formed per mg chloroplast protein per h). The problems of acetyl CoA synthesis in both compart­ments, chloroplasts and cytoplasm, are discussed in this context.

Vacuoles were isolated from the upper epidermis of petals of Petunia hybrida line R27 using three different procedures. Utilizing the vacuolar localization of anthocyanin, vacuolar prep­arations obtained through polybase treatment, osmotic shock and mechanical forces were com­pared on purity, yield and stability. The comparison indicated that application of the polybase procedure results in the best vacuolar preparations. Vacuoles could be obtained which main­tained their acidic pH and retained their anthocyanin content.

In vacuoles isolated from petal upper epidermis of Petunia hybrida through polybase-induced lysis of protoplasts the vacuolar localization of α-mannosidase and α-galactosidase was demon­strated. No association was observed between the tonoplast and these hydrolases. Protoplasts from different mutant lines blocked in anthocyanin biosynthesis were treated with DEAE-dextran to release vacuoles. The differences in quality of the obtained vacuolar preparations indicated the relation between mutant line used and successful application of the polybase-procedure.

From the culture medium of Pseudomonas sp. a further Fe containing complex, viz. dicyano- bis(pyridin-2,6-dicarbothioato)-ferrate (III) (2) has been isolated which participates in a ferrate (II)/ferrate (III) redox system (normal potential of-0.013 V) in the range of the redox potentials of cytochromes. Pyridine-2,6-di(monothiocarboxylic acid) which originally has been considered to be characteristic for Pseudomonas putida has been found recently as a metabolite of other bacterial strains (two of which have been characterized in this paper) as well.

We have studied in intact radish seedlings the effects of mevinolin (at concentrations of 0.25 to 5 μᴍ), a specific inhibitor of HMG-CoA reductase, and, therefore, of mevalonate biosynthesis, on the production of various isopentenoids and prenyllipids. Whereas the content of free desmethyl sterols was decreased steadily, only depending on the concentration of inhibitor present in the parts of seedlings investigated separately (e.g. roots, hypocotyls. and cotyledons), the effect on ubiquinone accumulation was different. Irrespective of the part of seedlings being analyzed, the maximal inhibition reached was 50%. Plastidic pigment accumulation, however, as well as that of chloroplast quinones (plastoquinone and phylloquinone), appeared even to be enhanced at low inhibitor concentrations and was not significantly lowered by application of 5 μᴍ mevinolin. α-Tocopherol showed a similar profile in the dose response to compounds known to be exclusively synthesized in the plastid. The results indicate a differential accessibility of the mevalonate synthesizing enzymes presumably present in the cytoplasm, mitochondria and plastids in respect to the inhibitory action of mevinolin. If prenyllipid formation in the different cell compartments solely depended on cytoplasmic mevalonate biosynthesis, all prenyllipids should be affected to the same extent as the sterols, which are exclusively synthesized by cytoplasmic enzymes.

The reactivity of the phytochrome chromophore and related tetrapyrroles towards ozone and tetranitromethane was investigated. Both oxidizing reagents cause bleaching of the main absorp­tion band of the pigment. The rate constants for this bleaching were determined under conditions of pseudo first order reaction kinetics. The rate constants for the reaction with ozone are similar for native phytochrome and for freely accessible tetrapyrroles (biliverdin, small chromopeptides from phytochrome) indicating that accessibility is not the limiting factor for the reaction with ozone. Under a variety of conditions, the P fr chromophore reacts by about 10% faster than the P r chromophore. This may reflect the true difference in reactivity. The rate constants for the reaction with tetranitromethane are much larger for biliverdin, bilirubin and small chromopeptides from phytochrome than for native phytochrome. The limiting factor for this reaction in native phytochrome therefore is the accessibility of the chromophore by the reagent. Previous conclusions on the difference in exposure of the tetrapyrrole chromophore in P r and P fr are confirmed.

The bipolar main tetraether lipid (MPL) of Thermoplasma acidophilum has been shown to form typical liquid expanded films at the air-water interface. The limiting molecular area at the collaps pressure is approximately Ac=73 Å 2 per molecule. Monopolar aiphytanyl diether lipids were found to occupy the same area at high surface pressure as MPL. Thus, it was concluded that in the monofilm only one of the two polar headgroups of the MPL molecules is hydrated, i.e. that the single MPL molecules arc oriented upright. The packing properties of MPT. in the monofilm are determined by the properties of the branched alkyl chains only; the polar head groups do not contribute to the space requirement in the film. The collaps pressure of the MPL film is approximately 39 mN m -1 at 8°C. At a surface pressure of π = 30 mN m -1 and 20 °C the film is stable for many hours.

Chemiluminescence (CL) that appears during oxidation of lecithin and ascorbate has been studied. A simple system consisting only of purified lecithin, which has one double bond, and ascorbate as a physiological reductant with a low redox potential, was used. The CL spectrum of lecithin contain a strong band lying in the near infrared, and three bands at 20 900 cm -1 , 17 700 cm -1 and 15 800 cm -1 , being characteristic of singlet molecular oxygen ( 1 O 2 ). The effect of 1 O 2 quenchers on both autooxidation processes has also been investigated. The obtained results indicate that the main emitter is the 1 O 2 . An addition of ascorbate to the system lecithin plus buffer causes a decrease of CL intensity. That is a result of stronger quenching properties of ascorbate and not due to efficiency of the generation of 1 O 2 .

Growth intensity of the green alga, Scenedesmus obliquus, was measured in autotrophic cultures, diluted once daily, between 20 and 30 °C in a light-dark cycle of 16 : 8 h at initial optical densities between 0.02 and 1.2. Arrhenius analyses of the results showed linear relationships be­tween growth intensity and temperature below the temperature optimum. The temperature effects on growth, activation energy, deactivation energy and normalized Q 10 values were signifi­cantly influenced by the amount of available light energy per unit biomass. The temperature dependence of nutrient-limited growth was not considered.

The influence of Mg 2+ ions on the secondary and tertiary structure of the RNA from bacteriophage MS2 was investigated by small-angle X-ray scattering and light scattering and by sedimentation experiments. The analysis of the outer part of the X-ray scattering curve obtained at low temperature in the absence of Mg 2+ yielded a cross-section radius of gyration of 0.88 nm and a mass per unit length of 1720 g mol -1 nm -1 . Very similar values for these parameters, which refer to the secondary structure of the RNA molecule, were also derived from the X-ray scattering curves obtained in the presence of different amounts of Mg 2+ (0.07 to 1 ions per nucleotide). On the contrary, the inner part of the X-ray scattering curves turned out to be highly dependent on the Mg 2+ concen­tration: the cross-section radius of gyration and the mass per unit length, which were determined from the scattering curves at small angles as parameters related to the tertiary structure of the RNA, amounted to 3.11 nm and 4000 g mol -1 nm -1 , respectively, in the absence of Mg 2+ and in­creased significantly upon raising the concentration of Mg 2+ . The increase of these structural parameters was found to be accompanied by a decrease of the overall radius of gyration (as revealed indirectly by X-ray scattering and directly by light scattering measurements) and by an increase of the sedimentation coefficient. The results from the investigations of the RNA at low temperature clearly establish the existence of double-stranded structures down to very low Mg 2+ concentrations as well as the occurrence of Mg 2+ induced changes of the tertiary structure. In the absence of Mg 2+ the RNA molecule appears to be a rodlike particle of about 300 nm length with a lateral diameter of about 9 to 11 nm. Upon addition of increasing amounts of Mg 2+ this extended structure tightens, eventually forming a coil-like particle. Previous X-ray scattering experiments with MS2 RNA at ionic strength 0.1 (Zipper, Folkhard and Clauwaert, FEBS Lett. 56, 283-287 (1975)) have already established the axial dimensions of such coil-like particles to be about 63, 31 and 14 nm. The results from supplementary X-ray scattering experiments performed at higher temperatures in the absence or presence of Mg 2+ clearly reflect the loss of secondary structure due to melting of the RNA. This loss is, however, less pronounced at higher concentrations of Mg 2+ .

The nucleotide sequence of the bovine 1.723 satellite DNA repeated unit was determined. The 680 bp long period of this satellite DNA does not show any significant sequence similarities with the known bovine satellite DNAs. Short repetitive sequences which are parts of 680 bp long repeated units do not form any orderly periodical structure. It seems, however, that the basic repeated unit of the 1.723 bovine satellite DNA has been formed by successive duplications of two, about 100 bp long sequences. The sequence divergence between different copies of the 680 bp repeated unit was also analyzed.

The rate constant for strand breakage in poly(U) after reaction with hydrogen atoms in deoxygenated aqueous solution has been determined to be k = 1.5 s -1 at pH = 4-5 and 24 °C. Dithiothreitol has been found to prevent strand break formation by reacting with H-adduct radicals of poly(U) with a rate constant of 5 × 10 6 M -1 s -1 . It is concluded that the rate-deter­mining step in H atom-induced strand breakage in poly(U) at pH ≦ 6 is the decay of uracil moiety H-adduct radicals via H-abstraction from the ribose moiety.

Helix-Lectins, Monoclonal Antibodies against Lectins, C om petition between Antibodies and Carbohydrates Monoclonal antibodies were raised against the lectin of H elix pom atia (HPL). Besides anti­ bodies bearing the more common y and x chains, antibodies with a, /j. and X2 chains were elicited. The anti-HPL antibodies are expected to be useful in studies on HPL biogenesis and HPL sub­ structure and in studies concerned with the binding of HPL to cell surfaces. Binding of carbohydrates to HPL im paired the binding of anti-H PL antibodies. One to 3 m M GalNAc inhibited HPL-binding in two out of nine antibodies. None of the antibodies bound in the presence of micrograms per ml of the polyvalent blood group A-substance from hog stomach. Similarly, all anti-HPL antibodies were prevented from binding if non-inhibitory concentrations of A-substance were supplem ented with GalNAc. Lectins from H elix aspersa (HAL) and H elix lucorum (H LL) differed from HPL in antigenic properties. Only one anti-HPL antibody each bound these lectins as well as HPL. Binding of lectins of Cepaea and Rapana was scarcely detectable. Most of the anti-HPL antibodies and the m ultivalent H PL-antigens formed precipitation lines in double diffusion tests. At least two antibodies (IgMs) did so with HLL but none with HAL. The possibility that antibodies were selected because of unknown interactions between HPL and the carbohydrate moieties of certain fractions of antibodies was excluded by raising the antibodies in the presence of tunicamycin to inhibit N-glycosylation. Monoclonal antibodies were raised against the lectin of Helix pomatia (HPL). Besides anti­bodies bearing the more common γ and ϰ chains, antibodies with α, μ and λ 2 chains were elicited. The anti-HPL antibodies are expected to be useful in studies on HPL biogenesis and HPL sub­structure and in studies concerned with the binding of HPL to cell surfaces. Binding of carbohydrates to HPL impaired the binding of anti-HPL antibodies. One to 3 mᴍ GalNAc inhibited HPL-binding in two out of nine antibodies. None of the antibodies bound in the presence of micrograms per ml of the polyvalent blood group A-substance from hog stomach. Similarly, all anti-HPL antibodies were prevented from binding if non-inhibitory concentrations of A-substance were supplemented with GalNAc. Lectins from Helix aspersa (HAL) and Helix lucorum (HLL) differed from HPL in antigenic properties. Only one anti-HPL antibody each bound these lectins as well as HPL. Binding of lectins of Cepaea and Rapana was scarcely detectable. Most of the anti-HPL antibodies and the multivalent HPL-antigens formed precipitation lines in double diffusion tests. At least two antibodies (IgMs) did so with HLL but none with HAL. The possibility that antibodies were selected because of unknown interactions between HPL and the carbohydrate moieties of certain fractions of antibodies was excluded by raising the antibodies in the presence of tunicamycin to inhibit N-glycosylation.

The sex pheromone of Ctenopseustis obliquana was found to contain (Z)-8-tetradecenyl acetate and (Z)-5-tetradecenyl acetate in approximately 4:1 ratio. No A 11-tetradecenyl compound was detected in C. obliquana, in contrast with the pheromones thus far reported from species of the tribe Archipini elsewhere in the world.

Planotortrix excessana was found to include moths of two distinct pheromone-types which were not mutually attractive. Tetradecyl acetate and (Z)-8-tetradecenyl acetate were identified as pheromone components in one, and two other tetradecenyl acetates, probably (Z)-5-and (Z)-7-tetradecenyl acetate, in the other. By contrast with other pheromones reported from the tribe Archipini,⊿11-tetradecenyl compounds were not found in either pheromone-type.

Lures for a monitoring system based on sex attractant trapping of Agrotis segetum males were elaborated for Israel and Germany. Various mixtures of (Z)-5-aecenyl acetate, (Z)-7-dodecenyl acetate, (Z)-9-dodecenyl acetate, (Z)-9-tetradecenyl acetate, decyl acetate and dodecyl acetate were tested in 20 different blends. From comparison of all the trap catches a four-component lure consisting of (Z)-5-decenyl, (Z)-7-dodecenyl, (Z)-9-tetradecenyl and dodecyl acetate is recommended for monitoring of A. segetum in both countries.

The membrane current vs. voltage dependence was measured in Limulus ventral nerve photo­receptors at various external Ca 2+ and Na + concentrations, using the voltage clamp technique. Lowering the external concentration of the divalent cations Ca 2+ and Mg + to < 1 μmol/l by adding EDTA causes 1)the light-induced transient conductance increase to disappear and 2)the reversal potential of the membrane current in the dark to shift to a positive value between + 10 and + 20 mV. This value is about the same as the (V rev J D ), reversal potential of the total light current under normal ionic conditions. If the external Na + is lowered to 50 mmol/1 (i.e. 10% of the normal concentration) simultaneously with the lowering of the divalent cation concentration described above, the light response is not abolished and V rev J D is shifted less. The extent of this antagonism depends on the sodium sub­stitute; it is stronger if choline is used instead of lithium. Lowering of sodium alone to 50 mmol/1, in a saline containing normal Ca 2+ and Mg 2+ concen­trations, does not change the membrane dark current vs. voltage curve and so V rev J D is not altered; V rev ⊿J L , the reversal potential of the light-induced current, however, is reduced by 10 mV (from +20 to +10 mV). This reduction in V rev ⊿J L can be accounted for by the reduction of the sodium gradient across the cell membrane. Raising the external Ca 2+ concentration to 40 or 100 mmol/l has no conspicuous effect on the membrane current vs. voltage dependence and the gating of the light-induced conductance in­crease. The results are consistent with our working hypothesis that the gating of the light-activated ion channels in Limulus photoreceptor is controlled by negative binding sites for which calcium- and sodium ions compete with antagonistic actions.

Hydrogen atoms from the radiolysis of water at pH 1.6 add to the 5,6-double bond of pyrimidines. The preferen­tial site of attack is the C(5) position (values in brackets) in the case of 6-methyluracil (87%), 1,3-dimethyluracil (71%), uracil (69%) and poly(U) (60%). This reaction yields a radical of reducing properties which can be monitored by its reaction with tetranitromethane in a pulse radiolysis experiment. In thymine (37%), thymidine (32%) and 1,3-dimethylthymine (25%) H-addition no longer pre­ferentially occurs at C(5), but addition is now mainly at C(6). Hydrogen abstraction from the methyl groups or the sugar moiety is negligible (≦ 5.5%). A comparison is made with literature values for the equivalent reactions of OH radicals.

The chloroplastic glutamine synthetase taken from mustard leaves shows a positive cooperative glutamate binding. The S 0 . 5 -value depends on the DTE concentration in the homogenization buffer. Normal glutamate satura­tion kinetics have been found with the root enzyme and the enzyme of etiolated cotyledons. These are not influ­enced by thiol compounds. The possible function of the allosteric behaviour of GS 2 is discussed.