Volume 36, Issue 5-6

A high performance liquid chromatographic method for analyzing phenolic acids on reverse phase C-18 is described. Twelve substituted benzoic and cinnamic acid derivatives as well as the parent acids were resolved in less than 25 min. Separation was achieved by using a step-wise gradient of three different solvent systems and by adding ammonium acetate for preventing intramolecular hydrogen bonding of the acids. The applicability of the described m ethod to crude plant extracts is demonstrated with Adenostoma fasciculatum, Adenostoma sparsifolium and Phyllospadix scouleri. The percent composition of the phenolic acids within the three extracts is given.

Two DNA fractions with different physico-chemical properties were obtained from calf thymus. On the basis of our results as well as some literature data we suppose that these are eu-and heterochromatin DNAs.

High Pressure, NMR, NAD +-Model, Activation Volume The activation volume ⊿V* for the rotation of the N -aralkylpyridinium moiety in a synthetic NAD + model was studied by high pressure HRNMR. From the pressure dependence of the proton spectra a negative ⊿V* of -3 cm 3 · mol -1 is derived. It is concluded from this result, that the rotation of the N -aralkylpyridinium ring occurs in a complex geared m otion and not in 180°-flips.

By use of 13 CNM R, GC and GC-MS analysis, 60 compounds, mainly mono-and sesquiterpenes, were identified structurally from the essential oil of Artemisia absinthium L. The specific appli­ cation of 13 CNMR spectroscopy enabled the structure elucidation of the components. Actually for the first time trans-sabinylacetate could be identified in the essential oil from Artemisia absinthium; it even represents a main component of the oil.

The six common anthocyanidins found in Gladiolus petals occur in four types of glycosilation: 3-glucoside, 3-rhamnoglucoside, 3,5-diglucoside, and 3-rhamnoglucoside-5-glucoside. The six monoglucosides appear in minute quantities, whereas any of the other 18 anthocyanins can serve as the major contributor to the coloration of Gladiolus petals. In high performance liquid chromatographic analyses of petal pigment composition of nine cultivars, it was found that the anthocyanins are grouped on the basis of the aglycon substitution. Thus, pelargonidin appears by itself (group I), cyanidin and peonidin constitute group II, and delphinidin, petunidin, and malvidin group III.

Maturing seeds of Sinapis alba were incubated with D-[U- 14 C]glucose, sodium [l- 14 C]acetate or m>>O[U- 14 C]inositol in order to prepare radioactively labelled phytic acid with high specific activity. Although each substrate was utilized for the biosynthesis of phytic acid, maximum incorporation of radioactivity into phytic acid was found with rayoinositol. Radiochemical purity of the [U- 14 C]phytic acid preparations was confirm ed by chromatographic techniques. Such preparations should be useful for the study of interaction of phytic acid with metal ions and proteins and may serve as substrate in the assay of phytase.

The distribution of flavonoid glycosides and pyrones are investigated for 3 Aniba species. The intraspecific variation of these compounds is shown for Aniba rosaeodora. 6-(3′,4′-methylendioxy-trans-styryl)-2-pyrone and 6-(3′,4′-dimethoxy-trans-styryl)-2-pyrone are seldom occuring natural compounds and were proved for A. permollis for the first time. The phylogenetic aspects are briefly discussed.

4-Coumarate: CoA ligase (CL) isolated from oat primary leaves shows two activity maxim a in 5 and 7 day old seedlings, the first of which corresponds with the highest flavone accumulation rate. In this plant stage most of L-phenylalanine ammonia-lyase (PAL) is located in the epidermal tissues, whereas CL dominates in mesophyll protoplasts and in the vascular elements. In the older leaves, in which PAL is only detectable in the epiderm is and in vascular elements, most of CL is located in the latter. The CL from different tissues varies significantly in substrate specificity. Investigations of intact chloroplasts from isolated protoplasts revealed that approximately half of the PAL activity can be extracted from this organelle, whereas there is no direct evidence for the existence of CL in plastids.

Glutamine synthetase was partly purified from Chlorella pyrenoidosa. This enzyme activity was stimulated by dithioerythritol and 2′-3′-dim ercaptopropanol (BAL). Full activation was obtained by thioredoxins isolated from the cyanobacterium Synechococcus 6301, from spinach and by heated Chlorella extract. The apparent K m for the purified thioredoxin B from Synechococcus 6301 was determined to be 0.022 (22 × 10 -9 M), suggesting a specific role for thioredoxins in regulating glutamine synthetase activity in this alga.

Chemotrophically grown cells of Rhodopseudomonas capsulata contain at least three different pyridine nucleotide dehydrogenases, i) a soluble, found in the supernatant (144000 × g) of cell free extracts, NADH-dependent, ii) a mem brane-bound, NADH-dependent, and iii) a soluble, found in the supernatant N AD PH dependent. The membrane-bound NADH dehydrogenase (E.C. 1.6.99.3) has been solubilized by sodium deoxycholate treatm ent of m em branes and purified 75 fold by column chrom atography on Sephadex G-150 and DEAE cellulose in the presence of sodium cholate. The native enzyme has an apparent molecular mass (M r ) of 97 000, containing polypeptides of M r of about 15 000. The pH optim um was at 7.5. The enzyme was specific for NADH. The Michaelis constant for NADH and DCIP were 4.0 and 63 μm, respectively. The enzyme was inactivated by FMN, riboflavin and NADH. In contrast, the soluble NADH-dehydrogenase (i) was activated by FMN.

In vivo control of glutamine synthetase (GS) in the facultative phototrophic bacterium Rhodopseudomonas sphaeroides was studied. The enzyme was partially purified from cells grown photosynthetically in ammonium-malate-medium. Its catalytic properties were characteristically changed by incubation with phosphodiesterase indicating an in vivo regulation by adenylylation/deadenylylation. Adenylylation states of GS were measured as a function of variations in the nitrogen source and the light intensity during photosynthetic growth.

In flower extracts of defined genotypes of Antirrhinum majus, two different hydroxylases were found catalysing the hydroxylation of naringenin and eriodictyol in the 3-position and of naringenin in the 3′-position. The 3-hydroxylase is a soluble enzyme and belongs according to its cofactor requirement to the 2-oxoglutarate-dependent dioxygenases. Investigations on different genotypes revealed a clear correlation between block of the anthocyanin pathway by recessive alleles of the gene inc and a complete lack of 3-hydroxylase activity. Chemogenetic studies on different genotypes suggested that the 3′-hydroxyl group of the B-ring of flavonoids is introduced at the stage of C 15 intermediates. The corresponding 3′-hydroxylase was found to be localized in the microsomal fraction and required NADPH as cofactor. In confirmation of the chemogenetic studies, a strict correlation was found between 3′-hydroxylase activity and the gene eos which is known to control the hydroxylation of flavones, flavonols and anthocyanins in the 3′-position. These results are similar to those previously obtained with Matthioia incana.

Bacteriochlorophyll b (bchl b) has been isolated from the halophilic photosynthetic bacterium, Ectothiorhodospira halochloris. The pigment and a series of derivatives thereof are different from Bchl b from Rhodopseudomonas viriais by HPLC analysis, but similar by uv-vis spectroscopy. The chromatographic difference originates in different esterifying alcohols in the two pigments. The one from Rp. viridis (Bchl b p ) is esterified with ⊿2-phytaenol (phytol), that from E. halochloris (Bchl b ⊿2,10 ) with ⊿2,10-phytadienol. The structure of the latter has been established by isolation of the alcohol from the purified pigment, followed by (i) gaschromatography-mass spectroscopy and (ii) ozonolysis and dinitrophenylhydrazon-formation of the cleavage products, which were identified by gaschromatography-mass spectroscopy as 6-methyl-heptan-2-one, and 4-methyl-nona-1,8-dione.

Illumination of 3 day old etiolated radish seedlings with continuous white light results in a progressive accumulation of chlorophyll a and b. Both pigments are bound in a different way to the thylakoid chlorophyll-proteins, which appear parallel to the formation of chlorophylls. By applying the SDS-PAGE method to SDS-digested chloroplasts, it was possible to show that the chloroplasts of radish cotyledons contain the typical chlorophyll proteins LHCP 1-3 , CPa, CPI and CPIa which have been found in other plants. Between LHCP 1 and CPI an additional chlorophyll protein is detected with the spectral properties of a LHCP; it is termed here LHCP y . When the green plants are transferred to continuous darkness, chlorophylls and the chlorophyll-proteins are progressively degraded. At an early stage of greening chlorophyll b is destroyed at a much higher rate in darkness than chlorophyll a, which yields high chlorophyll a/b ratios. This is paralleled by a faster decrease in the level of the corresponding chloro­phyll a/b-protein LHCP 3 than of CPI. At a later stage of greening, after the end of the logarithmic chlorophyll accumulation, the chlorophylls a and b and also the LHCP3 and CPI are destroyed in continuous darkness at equal rates; the a/b ratios and the LHCP 3 /CPI ratios are then little different from the light control. The data indicate that at an early stage of greening the light-harvesting chlorophyll a/b-protein LHCP 3 is less stable than the other chlorophyll-proteins (CPI, CPIa, CPa), which contain pre­ dominantly chlorophyll a. The ratio chlorophyll a to β-carotene (a/c ratio) of CPIa, CPI and CPa is about 10, while that of the LHCP 1-3 is found to be between 150 to 300. We therefore propose using the a/c ratio to define the chlorophyll-proteins which, besides the absorption spectra, is the most suitable parameter.

Quantitative absorption spectra in the visible and UV region were recorded for denaturated phytochrome (P r) and phytochromobilin peptides in comparison with native Pr. The method was tested with C-phycocyanin from Spirulina platensis. Based on known molar absorptivities for denaturated phycocyanin and suitable model compounds, and on the ratio A 620 native /A 655-665 degraded = 2.9, ^native was determined to be 102000 m -1 cm -1 for one phycocyanobilin chromophore in native phycocyanin. Likewise, Native was calculated to be 19000 M -1 cm -1 . The corresponding ratios for P r were A native /A denatured = 3.4 and A native /A degraded = 3.7, this yielded ε 665 native = 109000 to 118000 and ε 380 native = 36000 M -1 cm -1 for phytochromobilin in native P r. This value corresponds to one phytochromobilin per small phytochrome (60000 D) if data from the literature are corrected for the content of colorless proteins. The latter has been asessed from (i) the purity index (A 280 /A 665 ) and (ii) the contribution of the phytochromobilin chromophore at 280 nm as derived from model compounds.

Chromopeptides were prepared from the P fr form of phytochrome by pepsin digestion. After separation from colorless peptides and Pr peptides by column chromatography, spectral characteristics of this P fr peptide were determined (λ 1 max = 610nm, λ 2 max = 380nm in acid methanol). Irradiation of P fr peptide produces Pr peptide without liberation of a detectable compound. The P fr peptide is more sensitive to oxidation and reduction than the P r peptide. Oxidation with iodine and reduction with dithionite leads to partial chemoconversion of the P fr peptide to the P r peptide. The results favor the model of cis-trans isomerization for the Pr ⇌ Pfr transformation.

The Kok effect of photosynthesis was investigated in different tobacco mutants. It was found that the breaks in the light intensity curve were always at or around 1000 lux in all plants tested regardless of the unit sizes which differed by a factor of 10. It was concluded that the photo­ receptor responsible for the effect must be present in the wild type and the chlorophyll deficient mutants in the same amount and is probably not chlorophyll. Due to the fact that the light dependency of the Hill reaction in isolated tobacco chloroplasts also shows a break at or around the “Kok intensity” it was concluded that probably a structural change of the photochemical apparatus around 1000 lux contributes to the effect. Measurement of 180 2-uptake by mass spectrometry at low light intensity shows at low CO 2 -concentration an enhancement of 180 2-uptake again at/around 1000 lux indicating that photorespiration starts to function at the “Kok intensity”. Due to the fact that 180 2-uptake remains constant at high CO 2 -concentrations the break in the photosynthetic light intensity curve cannot be due to an inhibition of “dark respiration” at low light intensities.

Succulence-induction in Kalanchoe bloßfeldiana is demonstrated under long-day condition by application of a lipophilic fraction from flowering Kalanchoe. Different preparative TC separation steps yielded a partial purification of this fraction and the MS-identification of pterosterone (10 -6 m), which is not necessary correlated with the above described physiological activity.

By coupling 5-Methyl-cytidine to bovine serum albumin, a conjugate containing 18 mol of 5-Methyl-cytidine per mol of BSA was prepared. Antibodies to this conjugate were produced by immunization of rabbits. Specificity of the antibody was assesed by gel diffusion in agar containing excessive amounts of the carrier BSA. A slight cross-reactivity with cytidine was eliminated by adsorption on the cross-reacting antigen. The isolation of 7 S immunoglobulin from the total globulin fraction was accomplished by chromatography on DEAE Sephadex-A-50, and a method for the rapid quantitation of the antibodies showed that 12.7% of the IgG protein are monospecific against 5-Methyl-cytidine bovine serum albumine.

The concentration of substances with glucagon-like immunoreactivity (GLI) was determined in arterial blood plasma of rats up to 4 weeks after partial hepatectomy. 10 h following surgery a more than 18-fold increase in GLI + glucagon immunoreactivity could be observed. About 2/3 of the maximum activity seems to be of non pancreatic origin. The levels returned to normal about 22 h after surgery.

The sterilizing effect of azadirachtin, an insect growth regulator from the neem tree, Azadirachta indica A. Juss, was tested with mature females of Locusta migratoria. After a single injection of 10 μg into females between 2 and 13 days after emergence, about 60% died during the following four days and all lost weight in the range of about 50%. If the compound was injected into animals of an age between 2 and 10 days, no maturation of the terminal oocytes could be observed. Injection at an age between 10 and 13 days after adult emergence resulted in ovaries which contained almost mature oocytes. Most of the treated locusts had no oviposition and only traces of ecdysteroids were present in their ovaries, as quantified by radioimmunoassay. The inhibition of oogenesis and of ovarian ecdysteroid synthesis by azadirachtin is discussed on the basis of its interference with the neuroendocrine control of hormone synthesis.

The response of the homolateral “horizontal cells” (H-cells) of the third visual neuropile of the blowfly, Phoenicia sericata, has been investigated by intracellular recordings coupled with dye injections (Procion Yellow, cobaltous chloride). Responses were obtained from their terminal axon endings in the central protocerebrum. Under natural conditions, the horizontal cells respond not with graded potentials as reported previously, but with action potentials upon stimulation by regressive pattern motion within the contralateral receptive field. The stimulus-induced responses change from action potentials to graded signals within a few minutes after opening the head capsule. The possible causes of this change, namely hypoxic conditions and a consequent depletion of the ATP-supply, are consistent with the experimental data: a change in the resting potential as well a reduction of the spike amplitude. Furthermore, the possibility of two functionally different output regions of the H-cells is discussed, namely, (1) the terminal axon endings appear to conduct action potentials induced by the contralateral input (to the H-cells). Thus, these endings provide a solely contralateral, i.e. “monocular output”. Whereas (2) the axonal arborisation appears to conduct graded signals elicited by the ipsilateral input onto which the contralateral input is superimposed, thus providing a “binocular output”.

Both male and female Drosophila melanogaster possess systems which enable them to track other walking flies visually. Males use this system predominantly in courtship; females, when of sufficient age but still unmated, have been observed to track other flies, at rates of up to 18 bouts per hour. This behaviour is most conveniently studied in groups of females. Although similar to the courtship tracking of males, fem ale/female tracking lacks the “circling” component of courtship. The data suggest a functional relationship of female/female tracking to reproductive behaviour, although none has yet been identified. Bouts of tracking by females are most frequently terminated by the following female, especially when the target female remains motionless. Comparison of the tracking parameters of males and females has revealed differences in the velocity and pathlength of tracking bouts, and in the position of the fly with respect to the target. Males of Canton and Kapelle strains differ in the translatory component of their courtship tracking, but such a difference was not evident between females of these strains. Furthermore, tracking females do not allow their distance to the target to rise to that permissible in males, which suggests some sex-specificity in a part of the control system for tracking.

In petals of Silene alba plants of Armenian populations, vitexin 2″-O-xyloside was demonstrated to be present. The enzyme, catalyzing the transfer of the xylose moiety of UDP-xylose to vitexin, has its optimal activity at pH 7.8. Mg 2+ and Mn 2+ stimulated the reaction. The K m values were 2 × 10 -5 M for vitexin and 3 × 10 -5 M for UDP-xylose.

In green parts of Silene plants of the genotype gl R two methylated flavonoids were found: isoscoparin and isosco-parin 2″-O-rhamnoside. An enzyme has been demonstrated to catalyze the transfer of the methyl moiety of S-adenosyl methionine to iso-orientin and iso-orientin 2″-O-rhamnosi-de. Maximal activity takes place at pH 8.0 -8 .2 . Of the metal ions Mn 2+ , Mg 2+ , Ca 2+ , Co 2+ , Zn 2+ and Hg 2+ , only Co 2+ stimulated the reaction at conc. > 2 mM . For the methyla­ tion of isoorientin the K m values were 4 × 10 -6 M for S-ade-nosyl methionine and 0.32 × 10 -3 m for iso-orientin. When isoorientin 2″-O-rhamnoside was used as substrate the K m values were 5 × 10 -6 m for S-adenosylmethionine and 7 × 10 -6 M for iso-orientin 2″-O-rhamnoside.

From fruiting bodies of Gomphidius 2,2′,4,4′,5,5′-hexahydroxybiphenyl (7) and a red pigment, gomphilactone (1), have been isolated. The latter may be derived biogenetically from 1,2,4-trihydroxybenzene (6) via oxidative dimerization to 3,5′-dihydroxydibenzoquinone (8) followed by Postemak rearrangement.

Blood distribution in the systemic circulation of bony fishes is effected by a dualistic construction of the vasculature: A secondary arterial system originates from the well known (primary) arteries by multiple root-like shunt vessels. These secondary arteries and the vessels they supply should no longer be regarded as lymphatics. After an intensive search for unequivocally lymphatic vessels in fish, it appears doubtful whether there are any at all in this class of vertebrates.

The arrangement of the lateral line afferents of salamanders as revealed by transganglionic staining with horse­ radish peroxidase is described. Each lateral line organ is supplied by two fibers only. In the medulla these two afferent fibers run in separate fiber bundles. It is suggested, that only those fibers contacting lateral line sensory cells with the same polarity form together one bundle. Bundles formed by anterior or posterior lateral line afferents are also clearly separated. Beside the lateral line organs smaller pit organs are described. These organs are supplied by one afferent only which reveals an arrangement in the medulla different from that of the lateral line afferents. Based on anatomical facts, these small pit organs are considered to be electroreceptors. Centrifugally projecting neurons, most probably efferents, are described in the medulla.