Volume 52, Issue 11-12

Pseudomonas fluorescens, Pseudomonas putida, Pyoverdine, Siderophore, Bacterial Classification From Pseudom onas fluorescens 9AW and from Pseudomonas putida 9BW identical pyo-verdine-type siderophores were isolated and their structures were elucidated by spectroscopic methods and degradation studies. These novel compounds are of interest as they contain L-threo-β-hydroxy histidine in their peptide chains, an amino acid sofar encountered in nature only rarely. The co-occurence of the same pyoverdine in different Pseudom onas species and its significance for the classification is discussed.

The alkaloid composition of Hyoscyam us boveanus, H. desertorum, H. muticus and H. albus was investigated by capillary GLC and GLC-mass spectrometry. Altogether 39 alka­loids of the tropane and pyrrolidine types were identified. Several alkaloids were found in the different species for the first time, e.g., 22 alkaloids in H. boveanus, 13 in H. desertorum, 24 in H. muticus and 4 in H. albus. 3β-Hyoscyamine, dehydrohyoscyamine, phyllalbine, 3α-phenylacetyltropane, phenylacetylscopine as well as phygrine and 2,3-diacetonyl-N-methyl-pyrrolidine are apparently new for the genus Hyoscyamus. Alkaloid profiles and alkaloid contents of flowers, fruits, leaves, stems and roots were determined and their chemoecological functions discussed.

Purification of a soluble cytochrome c 6 from the unicellular green alga Scenedesmus obliquus by a simple and rapid method is described. The purification procedure includes ammonium sulfate precipitation and non-denaturating PAGE. The N-terminal sequence of the first 20 amino acids was determined and shows 85% similarity and 75% identity to the sequence of cytochrome c 6 from the green alga Monoraphidium braunii. The ferrocyto-chrome shows typical UV/VIS absorption peaks at 552.9, 521.9 and 415.7 nm. The apparent molecular mass was estimated to be 12 kD a by SDS-PAGE. EPR-spectroscopy at 20K shows resonances indicative for two distinct low-spin heme forms.

Methyl and ethyl cinnamate are aroma volatiles frequently occurring in fruits. Evidence was obtained that the enzymatic transfer of cinnamic acid to endogenous alcohols present in fruits (methanol, ethanol, 1-propanol) depended on energy-rich 1-O-glycosyl esters of cinnamic acid which served as acyl donor molecules. A putative 1-O-trans-cinnamoyl-β-ᴅ-gluco-pyranose; alcohol cinnamoyltransferase from cape gooseberry (Physalis peruviana L.) was active towards 1-O-trans-cinnamoyl-β-ᴅ-glucopyranose and 1-O-trans-cinnamoyl-β-ᴅ-gentiobiose. The enzyme was purified 290-fold by a protocol including ammonium sulphate precipi­tation, solubilization by Triton X-100, gel permeation and affinity chromatography on concanavalin A. The acidic glycoprotein (pI = 4.8) most probably is membrane bound. The distribution of alcohol cinnamoyltransferase activity in gel chromatography fractions suggests a native M r of 75,000. For 1-O-trans-cinnamoyl-β-ᴅ-glucopyranose, an apparent K m of 69 μm was determined. At pH > 6.0, non-enzymatic transesterification superposes the enzymatic transformation.

Victorin C, a host-specific toxin produced by Helm inthosporium victoriae, induced hydro-xycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase (HHT, EC 2.3.1) ac­tivity in oat leaves (Avena sativa L., a cultivar carrying Pc-2 gene). This enzyme activity catalyzes the final step of biosynthesis of oat phytoalexins, avenanthramides. The HHT activ­ity was detected after 12 h of victorin C application and reached to a maximum by 18 h. The induction of HHT was dose-dependent. All of the putative precursors of avenanthramides acted as substrates for HHT. These findings indicate that the accumulation of avenanthram ­ ides by victorin C treatment is due to induction of HHT. The enzyme activity showed highest specificity to 5-hydroxyanthranilate for the anthranyl moiety, while feruloyl-CoA was most effective for cinnamoyl moiety. HHT induced by victorin C showed significantly lower affinity for anthranilic acid relative to the enzyme induced by oligo-N-acetylchitooligosaccharides, another elicitor, suggesting that isozymes of HHT occur in this plant.

4-Pentadecylpyridine was isolated from plums infected by Taphrina pruni. This fungal metabolite has been tested for its effect on polyphenoloxidase, using the polyphenoloxidase-catalysed addition of L-proline to 4-methylcatechol in a chromogenic assay The resulting data prove that 4-pentadecylpyridine is a competitive inhibitor to polyphenoloxidase with an inhibitor constant of 3.70 ± 0.03×10 -4 м . Further comparative measurements have demon­strated that the effect is due to the pyridine moiety. Probably reduced polyphenoloxidase-activity decreases the toxicity of o-diphenols to the fungus and thus increases susceptibility of plums to the fungus Taphrina pruni.

α-Tocoquinone (α-TQ ) and α-tocopherol (α-TOC) which cannot substitute for plastoquinone-9 (PQ-A) as an electron acceptor from photosystem II (PS II), influence the oxygen evolution activity of thylakoid membranes under continuous illumination. In the presence of the herbicide DCMU and the protonophore FCCP which stimulate cyclic electron transport around PS II, α-TQ decreased oxygen evolution whereas α-TOC enhanced it. The effects are attributed to a stimulation or an inhibition of cyclic electron transport around PS II by α-TQ and α-TOC, respectively. Results of flash light experiments on PS II preparations show that both α-TQ and α-TOC increased the d-parameter which describes the transition probability from the S 3- to the S 0- state of the oxygen-evolving complex, although to a smaller extent when PQ-A is added alone to the preparations. The initial S-state distribution in darkadapted samples was changed only upon PQ-A addition and influenced neither by α-TQ nor by α-TO C supplementation. These effects indicate different kinds of interaction of PQ-A, α-TQ and α-TOC with the PS II components. α-TQ increased and α-TOC decreased the “total miss” parameter both in the presence or absence of PQ-A. A possible site of interaction of α-TQ and α-TO C with the cyclic electron transport around PS II is suggested.

The filamentous non-heterocystous cyanobacterium Oscillatoria chalybea is capable to photoevolve molecular hydrogen when the cells are flushed to anaerobiosis with nitrogen or argon and exposed to short light flashes or continuous light. The light-induced hydrogen gas exchange of Oscillatoria chalybea has been investigated by direct determination of dynamic changes in the hydrogen partial pressure at m/e=2 in the H/D collector of a mass spectrometric set-up. By means of this technique also the time curves of the light-induced hydrogen gas exchange could be directly recorded. Depending on the chlorophyll concentration in the measuring cell we observed an increasing hydrogen content of the aqueous Oscillatoria suspension i.e. a dark evolution of molecular hydrogen. Upon the onset of light an initial rise of the H 2 -signal was observed which was increasingly mixed or followed by a hydrogen uptake. The capability to photoevolve molecular hydrogen was maximal with young cultures and decreased with increasing age. The hydrogen evolution signals require relatively short dark adaptation to get pronounced; few seconds suffice for 2/3 of the hydrogen evolution amplitude. Prolonged dark adaptation maximizes the flash amplitudes. The hydrogen evolu­tion signals do not deactivate by low flash frequency Oscillatoria chalybea evolves molecular hydrogen following growth on nitrogen free or nitrate containing medium. Increase of the oxygen partial pressure of the assays completely abolishes the hydrogen evolution signals with an I 50 -value of 6 μm.

Trichloroacetate (TCA) affects the redox property of Y D (tyrosine-160 on D2 polypeptide) after the removal of 33 kDa extrinsic polypeptide from the photosystem II (PSII) core. However, TCA has no obvious effect on the redox property of Y Z , confirming that the environments around Y Z and Y D are quite different. The conclusion on the effects on Y D is based primarily on the observation that Y D + is not detected as the EPR signal II slow when 33 kDa polypeptide is released by TCA -treatment. Dialysis of the TCA -treated sample that allows the rebinding of mostly 33 kDa polypeptide restores Y D+, showing that the loss of the EPR signal II slow| takes place after the removal of 33 kD a polypeptide but not the release of manganese (Mn). Additionally, treatment of several halogenated acetates on Tris-washed PSII particles shows that the degree of their effects on suppressing EPR signal II slow is correlated with their hydrophobicity. It is postulated that Y D becomes more accessible to some small hydrophobic molecules depending upon their hydrophobicity when 33 kDa poly­peptide is removed.

Subunit I of chloroplast ATP synthase is reviewed until now to be equivalent to subunit b of Escherichia coli ATP synthase, whereas subunit II is suggested to be an additional subunit in photosynthetic ATP synthases lacking a counterpart in E. coli. After publication of some sequences of subunits II a revision of this assignment is necessary. Based on the analysis of 51 amino acid sequences of b-type subunits concerning similarities in primary structure, iso­electric point and a discovered discontinuous structural feature, our data provide evidence that chloroplast subunit II (subunit b' of photosynthetic eubacteria) and not chloroplast subunit I (subunit b of photosynthetic eubacteria) is the equivalent of subunit b of nonphoto­ synthetic eubacteria, and therefore does have a counterpart in e.g. E. coli. In consequence, structural features essential for function should be looked for on subunit II (b').

This study shows that incubation of rat liver mitochondria in the presence of the thiol/ amino reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DID S) is followed by inhibition of both succinate supported respiration and oxidative phosphorylation. Half-maximal inhibition of succinic dehydrogenase activity and succinate oxidation by mitochondria was attained at 55.3 and 60.8 μm DIDS, respectively. DIDS did inhibit the net ATP synthesis and ATP ⇔ [ 32 P]Pi exchange reaction catalyzed by submitochondrial particles in a dose-dependent manner (K i = 31.7 μm and K i = 32.7 μm), respectively. The hydrolytic activities of uncoupled heart submitochondrial particles and purified F 1 -ATPase were also inhibited 50% by 31.9 and 20.9 μm DIDS, respectively.

ADP-glucose pyrophosphorylase (AGPase), a two-gene-encoded enzyme, is the key com­ponent of starch synthesis in all plants. In the present study, we have used an E. coli expression system for the (over)production of proteins derived from both full length and specifically truncated cDNAs encoding small subunits of AGPase from seed endosperm (AG Pase-Bl) and leaves (AGPase-B2) of barley (Hordeum vulgare). Based on immunoblot analyses, the molecular mass of the expressed A G Pase-Bl (52 kD) was similar to that from endosperm extracts, whereas the expressed AGPase-B2 (56 kD) was larger than that in barley leaves (51 kD). Expression of truncated cDNAs for both the seed and leaf proteins has allowed for a direct verification of molecular masses that were earlier proposed for mature AGPases in barley tissues. The data suggest that seed AGPase-B1 does not undergo any post-translational proteolytic processing in barley, whereas the leaf homologue is processed to a smaller protein. Possible implications of these findings are discussed.

Using crude extracts from the cyanobacterium Anabaena variabilis glutamine synthetase (GS) activity was rapidly irreversibly reduced to about 60% during dark incubation ("sponta­neous GS inactivation"). An additional decrease was observed by the addition of ammonia in the light ("ammonia-mediated inactivation"). Both effects were prevented by EDTA, MnCl 2 or catalase indicative of the involvement of H 2 0 2 . This is a key intermediate in oxidative enzyme inactivation. In both spontaneous and am monia-mediated GS inactivation H 2 0 2 is produced in different ways. Spontaneous inactivation is prevented by depletion of reduced pyridine nucleotides which apparently donate electrons to produce H 2 0 2 . Fractionation of the crude extract showed that the light-enhanced GS inactivation by ammonia required the presence of thylakoid membranes. The photosynthesis inhibitor DCMU decreased GS inactivation by ammonia. For the inactivation in the light apparently H 2 0 2 is produced from super­ oxide during photosynthetic electron transport.

Trihydroxyflavones of Scutellaria baicalensis, Antioxidant Activity, Liposome, Peroxidation, MDA The antioxidant effect of a trihydroxyflavone extract from Scutellaria baicalensis on oxida­ tion induced by ultraviolet light, was studied with phosphatidylcholine liposome membrane. Also, as standards, the antioxidative activity of baicalin, wogonin, baicalein and butylated hydroxytoluene (BHT) was investigated. Comparison of the protective effects of thecom­pounds studied against photoinduced lipid peroxidation in lecithin liposome membranes showed that: (1) the inhibitory effect of those compounds (at 1.2 mol% antioxidant content in liposomes) on TBA reactive materials from lipid peroxidation decreased in the order of baicalin > BHT ≅ Scutellaria baicalensis. These were found much greater than wogonin and baicalein; (2) the depressed effect of those compounds (at 1.1 mol% compounds content in liposomes) on the production of conjugated dienes (proportional to oxidation index) could be classified as follows: Scutellaria baicalensis ≅ baicalin > BHT, these three were found more active much greather than baicalein and wogonin. Results obtained by ESR measure­ment confirm that Scutellaria baicalensis extract and the BHT compound significantly de­ pressed the effect of liposome oxidation. It was found that the new trihydroxyflavones of Scutellaria baicalensis, ensured a very satisfactory concentration-dependent protection of the liposome membrane against UV-induced oxidation. These findings suggest that some of the beneficial effects of the extract of the Scutellaria baicalensis can be mediated in certain diseases (for example in skin diseases) by their ability to scavenge free radicals and by their protective effect on lipid peroxidation caused by sunlight irradiation.

The gas exchange between the root system of the European alder (Alnus glutinosa (L.) Gaertn.) and the surrounding soil is studied using four-year-old trees. For the experiments the root system connected to its stump is exposed to gases of different molar mass. The stump is cut above the soil surface and in contact with the external atmosphere. A net convective volume flow of gas from the soil into the roots and out of the stump is observed if the mean molar mass of the gas present in the soil is lower than that present in the intercellular space of the roots. The direction of the convective gas flow can be reversed by increasing the molar mass of the gas present in the soil. These phenomena are governed by Graham 's law of diffusion. They demonstrate the importance of gas convection for the gas exchange in tree roots.

We evaluated free radical scavenging activity of the water, methanol and chloroform ex­tracts of propolis in 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and xanthine-xanthine oxidase (XOD) generated superoxide anion assay systems. The free radical scavenging activity guided fractionation and chemical analysis led to the isolation of a new compound, propol {3-[4-hydroxy-3-(3-oxo-but-1-enyl)-phenyl]-acrylic acid) from the water extract, which was more potent than most common antioxidants such as vitamin C and vitamin E (α-tocopherol) in these assay systems.

Lignans bind to sex hormone-binding globulin (SHBG ). The lignan with the highest binding affinity is (±)-3,4-divanillyltetrahydrofuran. In a double Stobbe condensation - without use of protecting groups - a wide variety of lignans with different substitution pattern in the aromatic and aliphatic part of the molecule was synthesized. These lignans were tested in a SHBG -binding assay which allowed to deduce the following relationship between structure and activity: 1) (±)-diastereoisomers are more active than meso compounds 2.) the 4-hydroxy- 3-methoxy (guajacyl) substitution pattern in the aromatic part is most effective 3.) the activity increases with the decline in polarity of the aliphatic part of the molecule.

The anti-inflammatory activity of andalusol (ent-13(16),14-labdadiene-6α,8α,18-triol), a diterpenoid obtained from the acetone extract of Sideritis foetens Clemen, has been investigated. This compound was able to inhibit acute inflammatory processes induced by carrageenan or 12-O -tetradecanoylphorbol acetate (TPA ) after oral or topical administration. Quantitation of the neutrophil specific marker, myeloperoxidase (MPO), demonstrated that its topical anti-inflammatory effect was associated with reduction in neutrophil infiltration into inflamed tissues. Interaction of andalusol with leukocyte functions and histamine release from mast cells was analyzed in vitro. At a concentration of 100 μm andalusol decreased β-glucuronidase release from calcium ionophore A23187-stimulated rat peritoneal leukocytes. However, it failed to affect superoxide generation on TPA -stimulated leukocytes and it was non toxic to leukocytes up to 100 μm (assayed in terms of lactate dehydrogenase release). Andalusol produced a dose-dependent inhibition on histamine release from rat peritoneal mast cells stimulated by com pound 48/80 or calcium ionophore A23187. These results suggest that andalusol possesses an anti-inflammatory profile, and it is in part responsible for the anti-inflammatory activity attributed to this plant.

The activity of thymidine phosphorylase (dThdPase) in the myometrium and uterine myomas has been investigated in perimenopausal women. Differences in the activity of dThdPase have been found depending on the myoma type, menopause stage and the phase of the menstrual cycle in which the surgery was performed. The enzyme in the cytoplasmatic soluble fraction obtain ed at 50 000 x g was the most active in cellular leiomyomas of the follicular phase, the least in adenomyom as of the luteal phase of the menstrual cycle, whereas its activity in myometrium was always unchanged. Greater differences can be observed in the activity of dThdPase after a partial purification of the enzyme from myomas. It seems that the increase in dThdPase activity may point to its correlation with transient, premalignant tumor which may later transform into malignant forms.

From the culture medium of Azomonas macrocytogenes a 5,6-dihydro-isopyoverdin could be isolated which includes the same peptide chain as the accompanying isopyoverdins (azoverdins). As analogous pairs had been encountered in the pyoverdin series where a dihydro derivatives are considered to be the immediate precursors of the pyoverdins, the same biogenetic sequence can thus be assumed for the iso-series.

A new valerenane sesquiterpenoid, methyl valerenate (1), was found in the essential oil from underground parts of Valeriana officinalis L. s.l. Spectral data of methyl valerenate, which was synthesized from valerenic acid (2), are given in this paper.