Volume 51, Issue 7-8

Three months old plants of the Chinese tung-oil tree Aleurites montana were cultivated for 4 months in air containing an increased amount of 700 ppm CO 2 . During the exposure to 700 ppm CO 2 the plants exhibited a considerably stronger growth (30-40% ) in comparison to the control plants (grown in normal air). In these CO 2 -plants during the entire analyzing period the amount of soluble proteins, of soluble sugars and the chlorophyll content were lower than in control plants. The protein content, referred to leaf area, increased during this time in both plant types by approx. 50% but with a different time course. The increase is faster in CO 2 -plants compared to control plants, and ends up with similar values in both plants after 4 months. No difference is seen between sun and shade leaves. The chlorophyll content in both sun and shade leaves is 20% lower in CO 2 -plants. Whereas the chlorophyll content in sun leaves stays constant during developm ent, it has increased in shade leaves by 20% at the end of the 4 months period. The content of soluble sugars is lower in CO 2 -plants compared to control plants. The difference is bigger in sun leaves than in shade leaves. The ribulose 1.5-bisphosphate carboxylase/oxygenase content almost doubles within the experimentation period, but seems to be subject to large variations. CO 2 -plants contain in general less ribulose 1.5-bisphosphate carboxylase/oxygenase than control plants. The content of coupling factor of photophosphorylation is 20% lower in CO 2 -plants when compared to control plants and remains during development more constant in CO 2 -plants. The molecular structure of the photosystem II-complex undergoes under the influence of the increased CO 2 -content a quantitative modification. The light harvesting complex (LHCP) and the extrinsic peptide with the molecular mass of 33 kDa increase in CO 2 -plants. Gassing with SO 2 (0.3 ppm in air) leads to a strong damage of the plants. The damaging influence is already seen after 6 days and leads to a partial leaf-shedding of the tree. In the visually still intact remaining leaves the chlorophyll content referred to unit leaf area decreases by 63%, that of soluble sugars by 65%, the content of soluble proteins and that of Rubisco decrease by 26% and 36% respectively. The light harvesting complex and the chlorophyll- binding peptides (43 and 47 kDa) increase whereas the extrinsic peptides decrease. It looks as if the simultaneous application of SO 2 (0.3 ppm) and increased CO 2 (700 ppm) releaves the damaging effect of SO 2 . Plant growth does not exhibit a difference in comparison to control plants. Soluble proteins and chlorophyll increase by 27% and 33% and the ribulose 1.5-bisphosphate carboxylase/oxygenase content as well as that of soluble sugars increases by 18 respectively 14%. The peptide composition of photosystem II shows a quantitative modification. The LHCP increases and the chlorophyll-binding peptides and the peptides with a molecular mass smaller than 24 kDa are reduced. The quantity of extrinsic peptides appears unchanged. Ribulose 1,5-bisphosphate carboxylase/oxygenase and the CF 1 -complex of Aleurites are immunochemically only partially identical to the corresponding enzymes of Nicotiana tabacum as demonstrated by tandem-cross-immune electrophoresis.

The major chlorophyll a/b-protein light-harvesting complex of photosystem II (LHCII) isolated form barley photosynthetic membrane was shown to contain five major polypeptides only two of which (26.7 and 25.6 kDa) were found to be its true constituents as judged by the ability to migrate as oligomers in various analytical systems. When analyzed by a vertical-bed non-denaturing isoelectric focusing the LHCII was resolved into five trimeric subcom­plexes (designated 1 -5 in order of decreasing p I) containing either only 26.7 kDa polypeptide (subcomplexes 1 and 2) or 26.7 and 25.6 kDa ones associated at 2:1 ratio (subcomplexes 3 -5) . The polypeptide of 26.7 kDa could be split by denaturing isoelectric focusing into fifteen molecular forms while nine molecular species were found to be constituents of 25.6 kDa polypeptide. The subcomplexes 1 -5 contained molecular forms of one or both polypep­ tides associated in sets of 7 -9 . Our findings favour the view that the apoproteins of LHCII are much more heterogenous than thought before.

The major light-harvesting chlorophyll a /b -protein complex of photosystem II (LHCII) from fresh barley leaves could be resolved by non-denaturing IEF into five trimeric subcomplexes designated 1 -5 in order of decrasing pi value. IEF-based analysis of PSII particles isolated from leaves in which the processes of senescence were induced by detachement and dark-incubation in the presence of water for 0 -8 days let us reveal that substantial rearrangements of LHCII organization took place throughout the course of senescence comprising a step-wise decline in relative abundance of subcomplexes 1 -3 (down to 0-58% of the initial abundance during 8 days of aging) and an increase in the relative abundance of the subcomplexes 4 and 5. Using SDS-PAGE and immunoblot analysis it was shown that the rearrangements were linked to the changes in the relative levels of LHCII apoproteins i.e. 26.7 and 25.6 kDa ones. The changes comprised the preferential disappearance of the 26.7 kDa polypeptide and an enrichment of 25.6 kD a one and most probably reflect the hetero­ geneity among LHCII apoproteins concerning their stability under the conditions of chi loss. Kinetin was able to repress the senescence-related rearrangements in LHCII subcomplex organization at late stages of aging (5 -8 days) by preventing over this time period the disappearance of 26.7 kD a polypeptide and the enrichement of 25.6 kDa one.

[U - 14 C]-Labelled 1,2,3,4,6-penta-O-galloyl-β-ᴅ-glucose was prepared by photoassimilation of 14 CO 2 with leaves from staghorn sumac (Rhus typhina) in the presence of the herbicide glyphosate. Extracts of the plant material were partitioned against ethyl acetate and chromatographed on Sephadex LH-20, yielding a series of crude tri-to decagalloylglucoses. The pentagalloylglucose fraction among these was further purified by HPLC to >99% purity and a specific radioactivity of 130 kBq (3.5 μCi) per μmol. The ratio of the radioactivities in the glucose and galloyl moieties, respectively, suggested a uniform labelling pattern of the product.

Dinucleoside 5′,5‴-P 1 ,P 4 -tetraphosphate hydrolase (EC 3.6.1.17) has been purified to homogeneity from tomato (Lycopersicon esculentum) cells grown in suspension. The purification procedure comprised ammonium sulphate fractionation following five standard chroma­ tography steps and a final chromatography on Ap 4 A-Sepharose. The homogeneous hydrolase has a molecular mass of 20 kD a and an isoelectric point of 4.5. The enzyme hydrolyses diadenosine tetraphosphate (Ap 4 A) asymmetrically to AMP and ATP. Among other naturally occurring dinucleoside oligophosphates, Ap 5 A and Ap 6 A are substrates whereas Ap 3 A is not. Of various phosphonate analogues tested, the Ap 5 A analogue, AppCH 2 pCH 2 ppA, was not cleaved and the Ap 3 A analogue, ApCH 2 CH 2 ppA. was a very poor substrate. Enzyme activity is stimulated by 5mм Mg 2+ and inhibited by fluoride anion; I 50 = 6.25 μм . The K m value for Ap 4 A is 0.8 μм . The enzyme exhibits a broad pH optimum from pH 6.5 to 9.0. In order to analyze the protein at the molecular level an internal peptide sequence from the homogeneous enzyme was identified. Within the sequence of 17 amino acids a kinase II motif as a general part of a conserved sequence of nucleotide binding sites was found. Against the internal peptide sequence a polyclonal antiserum was raised. By investigating the intracellular level of Ap 4 A hydrolase under different kinds of environmental stress, no changes occurred in response to heat shock. But, heavy metal stress and phosphate deprivation lead to a decrease in Ap 4 A hydrolase.

A new inhibitor of leukotriene biosynthesis, (+)-10α-hydroxy-4-muurolen-3-one (1), was isolated from fermentations of Favolaschia sp. 87129. Its structure was established by spectroscopic methods. The compound exhibited no antifungal or antibacterial activities. The effects of 1 on leukotriene biosynthesis were compared with (+)-T-cadinol, (-)-3-oxo-T-cadinol, and (+)-3a-hydroxy-T-cadinol, three related sesquiterpenes.

Three known sesquiterpenoids of the lactarane and secolactarane type, deoxylactarorufin A (1), blennin A (2) and blennin C (3), have been obtained from cultures of Lentinellus cochleatus (Basidiomycetes) together with the new metabolites (Z)-2-chloro-3-(4-me-thoxyphenyl)-2-propen-l-ol (4) and lentinellone (5), a protoilludane derivative. The structures were determined by spectroscopic investigations. 1, 2 and 3 are potent inhibitors of leukotriene biosynthesis in rat basophilic leukemia (RBL-1) cells and human peripheral blood leukocytes (PBL).

In the course of our search for new biologically active metabolites, lachnellin A (1), a metabolite with high cytotoxic and antimicrobial activities, the structurally related lachnellins B, C and D (3, 4, 7), and naphthalene-1,3,8-triol (8), an inhibitor of malate synthase (EC 4.1.3.2), were isolated from submerged cultures of the ascomycete Lachnellula sp. A 32 -8 9 . The antimicrobial, cytotoxic and phytotoxic activities of lachnellin A depended on its reactivity and could be abolished by the addition of cysteine. The enzyme inhibiting activity of (8) was due to reactive intermediates during melanization and was no longer observed in the presence of serum albumin. In addition, rac-scytalone (9), (+)-trans-3,4-dihydro-3,4,8-trihy-droxy-1(2H)-naphthalenone (10). 2,5-dihydroxytoluene (11), and (R)-(-)-5-methylmellein (12) were obtained from the same source and biologically characterized.

13-Hydroperoxy-9cis,12trans-octadecadienoic acid (13-LOOH) reacts with methionine and methionine-containing peptides in absence of any other reagent by slow release of ethylene. Ethylene was identified by mass spectrometry and quantified by gas chromatography. The amount of ethylene released in a certain time interval depends on the position of the methionine residue in the peptide chain. Highest rates of ethylene release were measured with peptides carrying methionine at the N-terminus. 13-Hydroperoxy-9cis,12trans-octadecadie-noic acid can be substituted by linoleic acid, lipoxigenase and oxygen. We assume that the instant formation of lipid peroxides after plant injury and the instant release of ′wound ethylene′ are related. Since the initiator tRNA in eucaryontic cells always carries a methionine, all newly produced proteins contain methionine at the N-terminal position and are therefore sensitive to oxidative damage by hydroperoxides of fatty acids.

A simple model thiadiazolidine, 5-(4-methoxycarbonylmethylthio-phenylimino)-3,4-tetra-methylene-1,3,4-thiadiazolidin-2-one, was synthesized and its structural modification investigated using glutathione S-transferase (GST) and an esterase preparation isolated from Echinochloa utilis. The objective is a better understanding of the metabolic activation of peroxidizing thiadiazolidine compounds. The model thiadiazolidine with an ester group (thiadiazolidine ester) was isomerized by GST to a more phytotoxic triazolidine structure (triazolidine ester). Both the thiadiazolidine and the more active triazolidine ester were hydrolyzed by Echinochloa esterase to less active free acid compounds. 5-(4-carboxymethylthiophenylimino)-3,4-tetramethylene-1,3,4-thiadiazolidin-2-one (thiadiazolidine acid) and 4-(4-carboxy-methylthiophenyl)-1,2-tetramethylene-1,2,4-triazolidin-3-one-5-thione (triazolidine acid), respectively. The thiadiazolidine acid, however, was only slightly converted into the triazolidine acid in the presence of GST. It is concluded that the thiadiazolidine ester was isomerized in Echinochloa to give the triazolidine acid through the triazolidine ester. Since the triazolidine ester exhibited the highest phytotoxic peroxidizing activity GST is considered as an activating enzyme for phytotoxicity and esterase as a detoxifying enzyme to reduce phytotoxic activity. Accordingly, phytotoxic thiadiazolidine-ester type herbicides may be produced by an interplay of isomerizing GST and esterase activity contributing to herbicide selectivity among plant species.

In the course of our study, it was found that phenyl propenoic acid derivatives were readily taken up by wheat. Leaf leachate components were chosen for the feeding experiments and p-coumaric acid, ferulic acid and caffeic acid were found to be quickly taken up into the plants via the roots. The analytical study revealed that the exudate contained potent antimicrobial compounds together with amino acids and sugars. Besides the primary metabolites, 4-hydroxystyrene, 3-methoxy-4-hydroxystyrene and 3-methoxy-4-acetoxystyrene were identified as exudate components from wheat roots in sterile hydroponic culture. This indicates that these antimicrobial components may play a significant role in the defense system as allelochemicals for the rhizosphere.

A non-radioactive cell-free assay was developed to quantitatively determine inhibition of plant-type phytoene desaturase by bleaching herbicides. An active desaturase was prepared from an appropriately cloned E. coli transformant. Another E. coli transformant was used to produce the required phytoene. Phytofluene and t-carotene, the products of the desaturase reaction, were either determined by HPLC or optical absorption spectra. Enzyme kinetics and inhibition data for the bleaching tetrazole herbicide WL110547 are presented as an example.

The effects of potassium-(picrate)-(18-crown-6) on the electron transport of photosystem II was investigated in isolated pea thylakoids. Low concentrations of the compound inhibited the fast decay of fluorescence yield associated with electron transfer between the primary (Q A ) and secondary (Q B ) quinone electron acceptor and increased the intermediary level of fluorescence to the F max level. The decay half-time of fluorescence yield measured in the presence of DCMU(S 2 Q A - charge recombination) decreased from about 1.8 s to about 0.3 s in thylakoids treated with potassium-(picrate)-(18-crown-6). While the inhibition of electron transport by DCMU gave rise to the appearance of a thermoluminescence band at about + 10°C (S 2 Q A - charge recombination) addition of potassium-(picrate)-(18-crown-6) resulted in a thermoluminescence band at about -10°C. Increasing concentrations of potassium-(picrate)-(18-crown-6) diminished the fluorescence yield and the -10°C TL band and abolished the Signal II S and Signal II f EPR signals of the tyrosine-D and tyrosine-Z electron donors, respectively. The phenolic-type inhibitor, potassium picrate had the same effect on thermoluminescence and on the tyrosine EPR signals. It is concluded that potassium-(picrate)-(18-crown-6) is a phenolic type inhibitor owing to its picrate constituent. At low concentrations picrate and potassium-(picrate)-18-crown) not only block the electron transport between Q A and Q B but they probably decrease the midpoint redox potential of Q A , as well. At high concentrations they also inhibit the light-induced oxidation of the tyrosine-D and tyrosine-Z donors.

Compounds extracted with methanol from the bulbs of Gladiolus spp. were tested for inhibition of Chl accumulation in greening cress (Lepidium sativum L.) seedlings. For the biotest, cress seeds were imbibed with water for 8 h and then incubated in the dark with the test solution for 40 h. Chlorophyll accumulation was determined at the end of the subsequent irradiation period of 8 h. Compounds active in this biotest were purified by several chromato­ graphic steps and identified by thin-layer chromatography with staining reactions and by gas chromatography/mass spectrometry. The following compounds were identified and their concentration for 50% inhibition of Chl accumulation determined: sucrose (75 mм) plus glucose (30 mм) , 3-hydroxybenzoic acid methylester (0.49 mм) and 5-hydroxymethylfurfural (6.7 mм) . The inhibitory action was compared with several substituted derivatives of the natural compounds.

Esculetin (6,7-dihydroxycoumarin) and its diacetate exhibited a marked inhibitory effect on Newcastle disease virus replication in cell cultures at concentrations of 36 jam and 62 jam, respectively. These compounds were selected from ten hydroxycoumarin derivatives through an in vitro antiviral screen involving viruses of the picorna-, orthomyxo-, paramyxo-, and herpes virus families.

Investigations were carried out to determine the antiulcerogenicity of the flavonoid fraction (ethyl acetate extract) of Erica andevalensis Cabezudo-Rivera on gastric ulceration induced by different experimental models. Oral treatment with the ethyl acetate extract and the major flavonoid (myricetin 3-O-ᴅ-galactoside) were found to be effective to prevent gastric ulceration induced by cold-restraint stress in rats . Statistically significant ulcer index values with respect to the control group were observed. Mucus content was not increased although it was accompanied by an increase in proteins and hexosamines. In pyloric-ligated animals flavonoids showed a significant reduction in the number and severity of the ulcers. Under the same conditions acidity did not decrease with the flavonic extract and myricetin 3-O-ᴅ-galactoside significantly as compared to control. Gastric ulcers induced by oral administration of absolute ethanol were reduced by pretreatment with the flavonoid extract of doses from 125 to 250 mg/kg and the isolated flavonoid of 25 mg/kg p.o. However neither the flavonic extract nor the isolated flavonoid induced changes in the amount and glycoprotein content of gastric mucus.

Absrtract The aim of this study was to elucidate the mechanism of the healing process mediated by the flavonic fraction of Bidens aurea on chronic gastric ulceration induced by 5% acetic acid in rats. The diethyl ether extract (125 and 62.5 mg kg- 1 body weight) was administered in a single doses, 7 and 14 days after provocation of lesions. Our results demonstrated that both doses significantly decreased the macro and microscopic ulcer index. Usually after 14-days treatment the lesions were found completely covered with regenerative epithelium and also an important proliferation of blood vessels was observed. Myeloperoxidase (MPO) activity was assayed and used as an index of leucocyte infiltration. Application of acetic acid produced a significant increase of this activity 7 days after induction of chronic injury. Administration of 125 mg kg -1 of the ether extract provoked a sharp reduction on the enzymatic activity at the same period. After 14 days, this decrease was higher with both doses (p<0.001). In addition, the macroscopic examination showed a drastic reduction of leucocyte infiltration in treated groups. These results suggest that the recovery of vascularization of the ulcerated area and the decrease of neutrophil infiltration are involved in the antiulcerogenic effect of the flavonoid fraction of Bidens aurea.

Binding and localization of the vasodilator and antitumor drug coactivator dipyridamole (DIP) and of its three derivatives, RA14. RA47 and RA25 (DIPD), to cationic (cetyltrimethylammonium chloride), anionic (sodium dodecylsulfate), zwitterionic (N-hexadecyl-N,N-di-methyl-3-ammonio-1-propanesulfonate), and neutral (t-octylphenoxypolyethoxyethanol) micelles was studied using fluorescence, optical absorption and 1 H NMR spectroscopy. The analysis of NMR, optical absorption and fluorescence data indicates that the depth of localization of the drugs in the micelles from the surface decreased in the order DIP > RA14 > RA47 > RA25. The binding constants for the neutral drug forms change in the same order in the range of 1400-3100 м -1 for DIP to 80-300 м -1 for RA25. This order is identical with the reported biological activity of DIPD. For the protonated drugs in zwitterionic or neutral micelles the binding constants are reduced by a factor of 20-75.

The glycogenolytic-sarcoplasmic reticulum complex from rat skeletal muscle accumulates Ca 2+ upon stimulation of glycogen phosphorolysis in the absence of added ATP. It is shown that an efficient Ca 2+ uptake involves the sequential action of glycogen phosphorylase, phosphoglucomutase and hexokinase, which generate low concentrations of ATP (approximately 1 -2 μм) compartmentalized in the immediate vicinity of the sarcoplasmic reticulum Ca 2+ , Mg 2+ -ATPase (the Ca 2+ pump). The Ca 2+ uptake supported by glycogenolysis in this subcellular structure is strongly stimulated by micromolar concentrations of AMP, showing that the glycogen phosphorylase associated with this complex is in the dephosphorylated b form. The results point out that the flux through this compartmentalized metabolic pathway should be enhanced in physiological conditions leading to increased AMP concentrations in the sarcoplasm, such as long-lasting contractions and in ischemic muscle.

The SPME (solid phase micro extraction) technique was used in the collection of volatiles released by calling females of the 4 -6 mm long tentiform leafminer moth Phyllonorycter sylvella (Lepidoptera, Gracillariidae). The volatiles released by the calling P. sylvella females were identified by GC-M S as a mixture of Z10-tetradecenyl acetate (92 %), E10-tetrad ecen yl acetate (2 %) and Z8 -tetradecenyl acetate (6 %). The amount of volatiles released by one calling female during three hours and collected on a polydimethylsiloxane fibre, was as large as the amount extracted from the glands of 20 females. The SPME technique gives the opportunity of continuously following the release of behaviour mediated signals from weak scented living organisms.

In a previous work a decrease in cholesterol and triglyceride plasma levels was observed in rats 24 hours after their exposure to a 12 Hz 6 mT pulsed magnetic field (PMF). This time, a study of intensity effects of a 12 Hz PMF for a sixty-minute exposure and of length of exposure for a 12 Hz 6 mT PMF took place. Non-linear effect-dose relationships were ob­ served for the PMF intensity as well as for the length of exposure used. The highest decreases in cholesterol and triglyceride levels were obtained after to a sixty-minute exposure with 1.5 mT and 12 mT.