Volume 45, Issue 3-4

Five kaempferol glycosides in the fronds of Asplenium scolopendrium were isolated . The structures of three novel flavonol glycosides were determined to be kaempferol 3-O -β-D -glucopyranosyl-( 1→3 )-β -D -(2 -O -caffeoyl)glucopyranoside 7 -O -α -L -rhamnopyranoside , kaempferol 3 -O -β -D -glucopyranosyl-(1→3 )-β -D -glucopyranoside 7 -O -α -L -rhamnopyranoside and kaempferol 3 -O -(2 -O -caffeoyl)-β -D -glucopyranoside 7 -O -α -L -rhamnopyranosideby means of spectral data (FAB-M S, 1 H- 1 H, 1 H - 13 C COSY and 1 H- 13 C long range COSY), respectively.

Further investigation of the isoflavonoids in white lupin roots has revealed a new coumaronochromone with a 2-hydroxy-3-methyl-3-butenyl side chain (lupinalbin G), three new pyrano-isoflavones (lupinisoflavones K and L, and allolicoisoflavone B) and two new di-hydrofurano-isoflavones each with a 2,3-dihydroxy-3-methylbutyl substituent (lupinisoflavones M and N). The biogenetic pathways for these and other lupin isoflavones are briefly discussed.

A further investigation of the root bark constituents of Jamaican dogwood (Piscidia erythrina) has revealed two new pyrano-rotenoids, (+)-erythynone and (+)-12a-hydroxyerythy-none. Both compounds co-occur with (-)-rotenone, and two additional rotenoids [(-)-12a -hydroxyrotenone and (-)-villosinol] not previously isolated from P. erythrina. Root extracts were also found to contain the rare isoflavone durmillone. The stereochemistry of all five Piscidia rotenoids was examined by ORD and CD spectrometry. These studies indicated that erythynone and 12 a-hydroxyerythynone were antipodal to naturally occurring (-) -6 aS; 12 aS-rotenone at the B/C ring junction.

The secondary product pattern of 38 specimens of Lasallia brigantium (Zsch.) Llano, L. hispanica (Frey) Sancho & Crespo and L. pustulata (L.) Merat were investigated by HPLC. A c­ cording to previous publications gyrophoric acid, lecanoric acid and umbilicaric acid were detected. Additionally two further unknown depsides were found. The presence of the anthraquinone skyrin, new to Lasallia, was shown for L. hispanica and L. pustulata. Skyrin is not equally distributed within the thalli but mainly concentrated in the umbilicus. In L. hispanica skyrin is located in the epihymenium, too. A correlation between secondary product pattern and thallus size or light supply does not exist.

A selection of lipids from achenes, cotyledons after germination, roots and leaves of normal and high oleic varieties of sunflower were analyzed with regard to their fatty acid profiles. The lipids included triacylglycerol and phosphatidylcholine as ER-made components and mono-and digalactosyl diacylglycerol as plastid-localized glycolipids. A comparison of fatty acid pat­ terns showed that the block in oleate desaturation of the high oleic variety is confined to the ER of fat accumulating embryos, but that upon germination the oleate desaturation in the cotyledonary ER is rapidly derepressed. These data are supported by enzymatic experiments. In microsomes from maturing fruits of the high oleic variety oleoyl-phosphatidylcholine desaturase could not be detected, whereas o leoyl-CoA : lyso-phosphatidylcholine acyltransferase and components of the microsomal electron transport chains were not affected. A correlation in the expression of desaturation blocks in seed and root fatty acids as observed in mutants of other species was not observed which, therefore, cannot be generalized. Our data are discussed in terms of the existence of two ER-specific oleate desaturase activities.

To establish the substrate specificity of the thioglucoside glucohydrolase myrosinase (EC 3.2.3.1), this enzyme was purified to homogeneity from light-grown cress (Lepidium sativum L.) seedlings by Sephadex gel filtration. Red Dye and anion exchange (FPLC Mono Q) chro­matography, and preparative isoelectric focusing. Hydrolytic activity was shown toward only 4 of the 29 synthetic and natural O-and S-glycosides tested. Highest activity was displayed with the endogenous glucosinolates benzylglucosinolate (K m , 295 μM) and sinigrin (K m , 300 μM) at an optimum pH of 5.5 in sodium citrate buffer. The synthetic glycosides PNPG (K m , 2.0 mM) and ONPG were poorer substrates at an optimum pH of 6.5 in potassium phos­phate buffer. The enzyme was inactive with all other nitrophenyl glycosides tested including PNP-a-D-glucoside and PNP-thio-β-D-glucoside, suggesting a requirement for O-β-D-glucose as the glycone moiety within these substrates. PNPG hydrolysis was stimulated 2.6-fold by ascorbate (1 mM). The enzyme exhibited no metal ion requirement and was strongly inhibited by lead nitrate, mercury chloride, and ferric chloride at 1 mM concentration. The metal chela­tors DIECA , EDTA , o-phenanthroline, and 2,2′-dipyridyl were not inhibitory, but the thiol reagents PCMS, PCMB, and N-ethylmaleimide (at 1mM) caused 50 -80% inhibition of enzyme activity.

Two antifungal metabolites isolated from Calcarisporium thermophilum were identified as 15-azahom osterols related to the compounds previously isolated from Geotrichum Jlavo-brun-neum. By full spectral comparison with authentic 15-aza-24-methylene-D-homocholesta-8,14-dien-3 β-ol (A 25822 B) from G. flavo-brunneum, the Calcarisporium metabolites were characterized as the 4a-m ethyl-and 4,4-dimethyl-analogues of A 25822 B. Several minor members of the series were also detected, and tentatively identified by MS analysis. The 15-azahom osterols exhibited good antifungal activity towards Candida parapsiliosis, though the activities were somewhat lower than that of the 4-demethyl derivative A 25822 B. Calcarisporium thermophi­lum is the second microorganism known to synthesize these unusual 15-azahomosterol deriva­tives.

The volatile shoot oil of Coleonema album was found to be composed of monoterpenes, ses­quiterpenes and phenylpropanoids. Heterotrophic and photom ixotrophic callus cultures were established from green parts of the plant. A photom ixotrophic cell line contained a heteroge­neous population of plastids and synthesized an essential oil, which differed qualitatively and quantitatively from the green parts of the intact plant. Substitution of 2,4-D and pCPA by a lower amount of NAA and high kinetin concentrations favoured the formation of essential oil. The heterotrophic callus, when grown on the same nutrient medium, was devoid of any differ­ entiated plastids and of essential oil constituents. Thus, a close correlation of light-induced plastid differentiation and essential oil synthesis was concluded.

Cyanoacrylates are potent inhibitors of photosynthetic electron transport (PET) and are potentially useful probes of the photosystem II herbicide binding site. A series of cyanoacrylates was synthesized and the Hill inhibition activities evaluated in order to select compounds suita­ble for radioactive synthesis. A cyanoacrylate, 2-(2-nitrophenoxy)ethyl 3-benzylamino-2-cyano-2-pentenoate, was found to displace diuron from the photosystem II herbicide binding site. For this compound the dissociation constant of the inhibitor/binding site complex was found to be 2 × 10- 8 M with an active site concentration of 2 nmol/mg chlorophyll. In a similar system the corresponding figures for diuron were 1.2 × 10 -8 m and 1.3 nmol/mg chlorophyll. Photoaffinity labelling of 1% II thylakoid proteins with 2-(2-azidophenoxy)ethyl 3-[7- 14 C]-benzylamino-2-cyano-2-pentenoate showed weak binding in the 32 and 28 kD mass regions, consistent with binding to the D, peptide.

The interior of chlorosomes, the main antenna system of the photosynthesizing bacterium Chloroßexus aurantiacus, is shown to contain no proteins in a fixed ratio to BChl c and in amounts that could be significant of direct chromophore-protein complexes. This excludes non-covalent chromophore-protein complexing -that has so far been found in all other antennae -as the main organizational principle of the interior architecture for chlorosom es of chlorophyll C. aurantiacus. Rather, these antennae constitute the first case of a chromophore-chromophore aggregate functioning as a photosynthetic light harvesting system.

Incubation of racemic ethyl [2,2- 2 H 2 ]-(E)-3,4-epoxydecanoate and the corresponding acid with intact cells of Sporobolomyces odorus led to the formation of deuterium labeled γ-decalaetone. The detection of labeled 2-decen-4-olide and 4-oxodecanoic acid made it possi­ble to propose the biogenetic sequence leading to γ-decalactone. The determination of the en­ antiomeric ratio of the lactone revealed an unspecific metabolism of the precursor. In contrast to the (E)-isomer, after administration of ethyl [5,6- 2 H 2 ]-(Z)-3,4-epoxydeeanoate no transfor­mation could be detected. These results indicate that (E)-3,4-epoxydecanoic acid, formed from (E)-3-decenoyl-CoA , an intermediate of the β-oxidation of linoleic acid, is the genuine precur­sor in the biosynthesis of γ-decalactone.

Oat mesophyll protoplasts were evacuolated by centrifugation on a Percoll gradient and used as starting material for establishing a functional cell-free system. For this purpose evacuolated protoplasts were osmotically lysed. From the resulting homogenate a cytosolic fraction was obtained by silicon oil filtration. This fraction was used to dilute preparations of lysed evacu­olated protoplasts in order to reduce the number of organelles per volume cytosol. The latter was necessary to increase light-dependent oxygen evolution of the cell-free system. The result­ing kind of “reconstituted” system showed light-dependent sucrose formation (about 100 nmol (mg Chi) -1 · hr -1 ) with bicarbonate as the only substrate. As this property depends on a functional interaction of chloroplast and cytosolic reactions, this cell-free system appeared to perform essential steps of partitioning of CO 2 between starch and sucrose. Addition of about 12 μm fructose 2 ,6 -bisphosphate, an inhibitor of fructose 1,6 -bisphosphatase and an activator of the PPi-dependent fructose 6 -phosphate phosphotransferase, caused sucrose degradation in the light. Thus, this cell-free system allows both the study of cytosolic enzyme activities under quasi in vivo conditions and the manipulation of cellular reaction sequences by plasma mem­brane-impermeable compounds.

The complete amino acid sequences of the hemoglobins from the adult European mink (Mustela lutreola) are presented. The erythrocytes contain two hemoglobin components and three globin chains. The isolation of globin chains achieved by ion-exchange chromatography on a column of CM -cellulose in 8 M urea buffer. The primary structure of globin chains and of the tryptic peptides determined in liquid-and gas-phase sequenators. The alignment of the a-and β-chains with those of reported sequences from other carnivora species belonging to the family Mustelidae may give an insight into the evolution of this molecule.

Entamoeba histolytica Entamoeba histolytica is a primitive eukaryote that lacks mitochondria, Golgi and a well-developed endoplasmic reticulum. Close to half of the cell volume is occupied by pinocytic vesicles, which are in continuous turnover with the plasma membrane and perform functions that in higher eukaryotic cells are taken over by lysosomes. Similar to the latter, the amebal vesicles are acidified. We report here that bafilomycin A I , a specific inhibitor of vacuolar-type (V-) ATPases, suppressed this acidification at submicromolar concentrations; concom itantly, it inhibited pinocytosis. These results strongly suggest the presence of a V-ATPase in pinocytic vesicles of E. histolytica, and thereby support the notion that the V-ATPases in the organelles of higher eukaryotes are derived from an archaic plasma membrane-bound form.

β-1,3-Glucanase and chitinase activities have been analyzed in cell suspension cultures of an A scochyta rabiei resistant ILC 3279 and a susceptible ILC 1929 cultivar of chickpea (Cicer arietinum) following treatment with an elicitor derived from this fungal pathogen. A facile method to determine both hydrolase activities in the cell culture medium was established. Significantly higher constitutive and elicitor-induced levels of both hydrolase activities were found in the medium of the ILC 3279 cell culture. The release of these enzyme activities is not due to cell lysis, but rather the consequence of a secretion mechanism. The cells of the resistant line contained a 5 times higher level of chitinase activity in comparison to the ILC 1929 cell culture, whereas the latter cells possessed a 3 times higher β -1,3-glucanase activity. The results are interpreted that accumulation of extracellular hydrolase activities may play an important role among the various plant defense mechanisms previously determined for the incompatible interaction between the resistant cultivar and its fungal pathogen.

The β-glucanase gene (bgl) from Bacillus amyloliquefaciens was expressed in E. coli CSH 55 under the control of the P R promoter of phage λ that is repressed by the thermosensitive repressor C 1857 . Production of β-glucanase was drastically stimulated by a temperature shift to 42 C. This overexpression of the bgl gene (about 20% of the total cellular protein) led to an almost complete excretion of the otherwise periplasmic protein into the extracellular medium, β-glucanase accounted for more than 50% of the extracellular proteins. Col E 1 related plasmid (pEG 1) are amplified in E. coli relA strains in response to an amino acid limitation leading to a 10-fold increase in the activity of plasmid encoded genes. In this work we intended to maximize the expression of the bgl gene by a concerted action of a plasmid amplification and temperature induction. Surprisingly we could not increase the β-glucanase production above the level reached by plasmid amplification or temperature in­duction alone. The reasons for this unexpected result will be discussed. Under all conditions tested the expression of the bgl gene was much lower in the E. coli rel A strain NF 162 than in E. coli CSH 55; the low β-glucanase production was accompanied by a reduced excretion rate of the enzyme.

Cells of Petroselinum crispum cv. “Paramount” and “Plain-leaved” immobilized in polyurethane foam retained the biotransformational characteristics of their freely suspended cells for certain monoterpenic substrates. Immobilized cells of both cultivars were more efficient than their free cells in isomerizing geraniol into nerol, but less efficient (<60% ) in reducing the aldehydes, citral and citronellal. Maintenance of the immobilized cells under the condition of growth limitation did not enhance the efficiency of the biochemical reactions. Under the same condition, the freely suspended cells of cult. “Paramount” exhibited higher efficiency (up to 1.6 times) of biotransformation of citral when compared to those in sucrose-supplemented media. This investigation lends support to the view that the biochemical productivity of cultured plant cells for especially monoterpenoids may not necessarily be improved upon immobilization.

The location of malate dehydrogenase in the cyanobacteria, Anabaena cylindrica, Nostoc muscorum and Chlorogloeopsis fritschii was investigated by the fractionation of cell-free extracts. The bulk of the enzyme activity was associated with the thylakoid membrane fraction, which also exhibited complete photosynthetic electron transport reactions. Malate dehydrogenase activity and photosystem II activities were inhibited by homologous antisera raised against isolated thylakoid membranes.

Changes in membrane polypeptide composition during greening of etiolated maize were investigated to confirm the existence of the developmental polypeptides of 12 - 15 kDa described recently in virescent soybean mutant [M. Droppa, M. L. Ghirardi, G. Horváth, and A. Melis, Biochim. Biophys. Acta 932, 138 - 145 (1988)]. These low molecular weight polypeptides were the most abundant proteins at the early stage of greening, but were largely absent from fully developed thylakoids. During greening the relative concentration of the 12-15 kDa polypeptides were inversely proportional to that of LHC II, suggesting a role of these polypeptides in the assembly of the LHC II and/or chloroplast development.

Illumination of thylakoid membranes in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea (DCMU) causes the reduction of the primary quinone acceptor QA of photosystem II (PS II) and the storage of a positive charge on the donor side of the photochemical reaction center. These oxidation-reduction reactions are accompanied by characteristic changes of absorbance in the ultra-violet region of the spectrum. The PS II-related absorbance difference spectra (250 -350 nm) were compared in control and hydroxylamine-treated thylakoid membranes, and in thylakoids suspended in the presence of carbonyl cyanide-p-(trifluoromethoxy)- phenylhydrazone (FCCP). The light minus dark difference spectra were dominated by the Q - A minus Q A difference spectrum. Qualitatively, the three spectra were identical in the 300 - 350 nm region, however, they showed distinct differences in the 250 - 300 nm region. The latter arose because of different contributions from the donor side of PS II in the thylakoid membrane of the three samples. The result suggested that FCCP acts as the ultimate electron donor in DCMU - poisoned chloroplasts. Therefore, the absorbance difference spectrum in the presence of FCCP reflected a contribution from the Q - A minus Q A component only. Deconvolution of the absorbance difference spectra of control and hydroxylamine-treated thylakoids yielded difference spectra attributed to the oxidation of a component on the donor side of PS II. This component did not conform with the known Mn(III) → Mn(IV) transition. Rather, it indicated the oxidation of a modified form of Mn in the presence of DCMU , probably a Mn(II) → Mn(III) transition. The results are discussed in terms of the use of DCMU - poisoned thylakoid membranes in the quantitation of the primary quinone acceptor Q A by spectrophotometric approaches.

Pentachlorophenol (PCP) is a widely used and highly toxic fungicide. Its toxicity is mainly expressed at the cell membrane level. It is, therefore, of interest to test its ability to alter the lipid bilayer organization. The present study was performed by X-ray diffraction techniques on dimyristoylphosphatidylethanolamine (DMPE) and dimyristoylphosphatidylcholine (DMPC) bilayers and by fluorescence on DMPC liposomes. These two phospholipids are respectively found at the inner and outer monolayers of human erythrocyte membranes. Each type of phospholipid was made to interact with different concentrations of the sodium form of PCP in absence and in presence of water. It was found that PCP significatively affected the structure of both phospholipids, being the damage much higher in DM PC bilayers.

The light-induced enhancement of the 7-ethoxycoumarin-O-deethylase activity was measured in a reconstituted system, consisting of the enzyme P-450 PB-B and the NADPH -cytochrome P-450 reductase. The relative increase of the activity was about 15%. It is shown that the product release process is accelerated by light. The phases of the catalytic cycle of 2 x 1012 protein complexes were locked by periodic application of light pulses (0.1 s duration, 1.32 s repetition time, and 390 -470 nm, 0.27 joule/nmol P-450). More than 80% of the active reconstituted enzyme complexes (= “molecular machines”) worked in phase after a few light pulses. The phase relation continued even after switching off the light pulses. The catalytic cycle time was 1.54 s, giving a turnover number of 39 min -1 . The turnover number, as determined from the enzyme activity under optimum conditions, was 39 min -1 . Due to the dissociation constant of the P-450 PB-B :NADPH - P-450 reductase complexes [3] only 24% of the proteins were in the active (= working) state under the conditions used. The lifetime of this complexes is larger than 6 s since more than 4 cycles of the free running enzyme can be observed. This is the first report, that all catalytic active complexes in the test tube can be synchronized by an external light source, if the right repetition time of the pulses is chosen, so that all these “molecular machines” work in phase.

In the course of the odontogenesis of bovine incisors several clearly distinguishable phosphohydrolase activities are observed in the pulp and in dental hard tissues. Using various substrates and inhibitors, unspecific alkaline phosphatase, two isoenzymes of acid phosphatase, Ca 2+ -activated ATPase and inorganic pyrophosphatase are characterized. The enzymatic activity of alkaline phosphatase in pulp and hard tissues is significantly high at the beginning of dentine and enamel mineralization. The specific activity of this enzyme decreases quite fast with the beginning of root formation, then more slowly, until it reaches a constant final value. Histochemical studies show that during mineralization the maximum of alkaline phosphatase activity is in the subodontoblasts. Lower enzyme concentrations are found in the stratum intermedium and in the outer enamel epithelium during that process. The specific activities of ATPase, acid phosphatases and pyrophosphatase show little temporal variation during tooth development, but they also appear in a characteristic spatial pattern in the dental tissues.

Chemical and enzymatic procedures are described for the synthesis of the monophosphates and cyclic phosphates of the antiviral acyclonucleoside 9-(1,3-dihydroxy-2-propoxym ethyl)-guanine (DHPG), its 3-hydroxymethyl-4-hydroxybutyl analogue, the (/?)-and (S)-epimers of 9-(3,4-dihydroxybutyl)guanine, and 9-(2,3-dihydroxypropyl)guanine. The structures, and some conformational features, of all the foregoing, were determined by 1 H and 31 PNMR spectroscopy. Their substrate/inhibitor properties have been examined in several enzyme systems, including ribonucleases, snake venom phosphodiesterase, beef heart and higher plant cyclic nucleotide phosphodiesterases, nuclease P1, and 3′-and 5′-nucleo-tidases. The enzymatic results are considered in relation to the mechanism of the antiviral ac­tivity of the cyclic phosphate of DHPG .

In a double-logarithmic plot of the rate of actin polymerization against monomer concentration, the slope has a lower value in the presence of the fungal metabolites cytochalasin B and D than in their absence. This suggests that actin oligomers which serve as polymerization nuclei are smaller in the presence of cytochalasins than in their absence.

We have investigated the influence of strong dc. magnetic fields - ranging up to 10 Tesla - on the weak sinusoidal electric signals which the electro-fish Apteronotus is known to emit. We made the following observations: 1. The amplitude of the sinusoidal electric signals rises with the application of the dc. magnetic fields, increasing by about 8% in a field of 10 Tesla. 2. The frequency of the signals of about 570 Hz was not at all affected by the field but remained constant in fields up to 10 Tesla. Since the electric signals of Apteronotus are of neural origin our experiments indicate an influence of strong dc. magnetic fields on the nervous system of this electric fish.

The galvanotropic response of the mycelial fungus Neurospora crassa is investigated. The angle distribution function of growing hyphae is described by a generating function which contains two non-trivial terms; one for directional growth and one for bidirectional growth. The following results were obtained, (i) Germ tubes grew towards the anode, (ii) The cellular response was linear for small sized cells and weaker electric fields. The galvanotropic constant, K GROW , which describes the linear response, was small for short germ tubes or hyphae (A GROW -1 = -20 V -cm -1 for I 0 = 10 |am) and large for longer cells (A GROW -1 = -1 -7 V ·cm -1 for I 0 = 100 μm). The growth coefficient K P of (-18.5 mV) describes the response independent of cell size. The linear response is explained by the field-induced distribution of charged membrane- bound proteins essential for galvanotropism. (iii) For E > E 0 = 3.8 V ·cm -1 , the linear response is inhibited (inhibition coefficient K 1 = 1.11). The inhibition is explained by field-induced changes of the membrane potential, (iv) The galvanotropic response of longer hyphae was bidirectional. The cells grew on average perpendicular to the applied field. The bidirectional response is proportional to Er with a bidirectional growth coefficient K 2 of -(0.25 V) -2 . The bidirectional growth is explained by the inhibition of the directed growth process. The transition from anode-directed to bidirectional growth was a function of the applied electric field as well as of the tube length (directed growth for E 2 -l 0 < 4.4V 2 ·cm -1 and bidirectional growth for E 2 ·l 0 > 4.4 V 2 · cm -1 ).