Volume 53, Issue 9-10

Supercritical fluid extraction (solvent: CO 2 and propane) was used for mild recovery of fatty oil of mille thistle fruit (Silybum marianum L. Gaertner). Fatty acid compounds (palmic acid, oleic acid, linolic acid, linolenic acid, behenic acid), coloring content (expressed in pheophytin and carotene), tocopherol content and concentrations of some metals (Al, As, B, Ba, Ca, Cd,Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, Mo, Na, Ni, Pb, S, Ti, V, Zn), boron and sulfur were determined from oils and compared with oil obtained by Soxhlet extraction. There is significant difference between supercritical oil and traditional oil in the quantity of bioactive compounds and elements. The ratio of bioactive components and elements of oils changed during the extraction. Supercritical oils have higher concentration of C18:1 oleic acid, C18:3 linolenic acid, C20:0 and C22:0 behenic acid than the oil gained by Soxhlet extraction. The oil obtained by supercritical fluid extraction at 80 bar and 25 °C with propane has the highest coloring content. Our paper is the first report to be published on element concentrations of mille thistle oils. Accumulation of some heavy metals can be observed in almost each oil sample.

Mineral composition, vitamins (group B, C and D), polyphenols, purine alkaloids, sugars and phytic acid were determined in twelve samples of cocoa husk of the main varieties of cocoa beans ( Theobroma cacao L.) cultivated in Africa and South America. Macro- (K, Mg, Ca, Na, P, and Na) and micro elements (Cu, Zn, Fe, and Cr) were quantified using an inductively coupled argon plasma-optical emission spectrometer (ICP-OES), on dry-ashed samples. Vitamins, phenolic compounds, alkaloids, sugars, and phytic acid were determined using HPLC. The mineral and vitamins intake that cocoa husk consumption could represent was evaluated with regard to the Recommended Dietary Allowances (RDA). The percentage of phytic acid is medium (0.98±0.13 g/100 g). The most abundant phenolic compounds found were caffeic acid and gentisic acid (2,5-dihydroxybenzoic acid).

2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and its glucoside, DIMBOA - G, appeared concurrently with germination in maize (Zea mays L.), and their concentrations per fresh weight reached a maximum 24-36 hr after germination. The aglycone then decreased to disappear as the plants began autotrophic growth, and the glucoside to a concentration half that of the maximum. The level of DIMBOA-G was always higher than that of the aglycone. A β-glucosidase activity which hydrolyzes DIMBOA-G to DIMBOA and glucose was detected in crude enzyme extracts from the seedlings. Its activity per fresh weight varied concurrently with the occurrence of DIMBOA. The purification was performed to an apparent homogeneity by cryoprecipitation followed by cation exchange chromatography and gel filtration. The K m value for DIMBOA-G was 0.07 mᴍ, whereas that for DIBOA-G, that was substantially absent in the seedlings of the cultivar studied, was 0.52 mᴍ. The activity on salicin and esculin was too low to be detected. The set of results suggested that free DIMBOA occurs constitutively or in a programmed manner as defense compound at a seedling stage of growth, and a specific glucosidase is involved in this process.

Plums (Prunus domestica) affected by the fungus Taphrina pruni and healthy ones were harvested in intervals of about four days. Photometric comparison of their methanolic extracts proved that infected fruits contained ten times more compounds with phenolic hydroxyl groups. Further structure elucidation and quantification of these phenolic differences by gas chromatography and gas chromatography / mass spectrometry revealed that the content of caffeoyl-ᴅ-quinic acid isomers and (+)-catechin had changed: The amount of chlorogenic acid and its isomers was increased in infected fruits about 15 times compared to nonaffected ones. Contrary, (+)-catechin content was decreased. Additional photometric assays demonstrated that (+)-catechin reduction is accompanied by a corresponding increase of proanthocyanidins in infected fruits. All the compounds identified in infected plums in increased concentrations had a common structural feature: they were o-diphenols. After oxidation to corresponding o-quinones they are able to add to substances with active hydrogen atoms, e.g. fungal enzymes. Consequently, the accumulation of a high concentration of odiphenols may be a defence response directed towards fungal enzymes.

Gibberella pulicaris is a causal agent of potato dry rot. The fungus is able to metabolize the potato phytoalexin rishitin, a trait which is possibly associated with virulence against potato tubers. Metabolism of the plant defence compound on agar medium is completed within 24 h. In contrast, incubations in various liquid media and buffers highly reduced degradation of rishitin with a maximal reduction of substrate down to 30% of the initial concentration within five days. The structurally related sesquiterpene lubimin was degraded completely within 12 hr in all tested liquid media. Our data suggest that rishitin metabolism is under an unusual genetic control requiring growth on a solid surface for efficient metabolism.

Stress, pathogens or insects induce profound changes in the expression of the chemical constituents of plants. Some of these changes could be adaptations to the new conditions or the expression of new defensive characteristics. The purpose of this paper was to analyze the effect of aphid infestation on the composition of endogenous compound, in barley leaves. Leaf waxes were obtained by washing with dichloromethane and remaining compounds were extracted with ethyl acetate and ethanol from homogenized leaves. The identification of compounds was done by combined capillary gas chromatography and mass spectrometry. Epicuticular compounds detected in barley after 6 days of aphid infestation were as follows (%): (1) 1-docosene (1.98), (2) 1-octadecene (2.07), (3) phytol (0.81). Epicuticular compounds in non-infested plants were (4) isophytol (0.83), (5) N-cyclohexyl-cyclohexanamine, (0.11) (6) 5-pentadecyl-1,3-benzenediol (2.10). In ethyl acetate extracts from infested plants the main compounds were: (1) (2.02), (7) 2,5-diethylpyrazine (0.61), (8) (5E)-eicosene (1.61), (9) (3E)-eicosene (1.93), (10) (9E)-eicosene (1.80). In non-infested plants the only detected compounds were (1) (1.02), (6) (1.91), and (7) (0.43). In methanol extracts from infested plants the following compounds were identified: (6) (2.15), (7) (0.21), (11) 2,2,6.6 tetramethyl- piperidine (0.12), (12) indole (1.01), (13) heptadecane (0.10), (14) methyl-β-ᴅ-glucopyranoside (0.21), (15) 2,5- dihydroxybenzaldehyde (0.27) and (16) N-phenyl-2-naphthalenamine (0.29). In polar extracts from non-infested plants the most abundant compounds were (4) (1.40), (7) (0.18), (17) (9Z,12Z,15Z)-octadecatrienoic methyl ester (1.01), (18) (9Z,15Z)- octadienoic methyl ester (2.01), and (19) hexadecanoic methyl ester (1.93). Compounds 4, 5 and 7 -1 9 are described for the first time in Hordeum vulgare.

Axenic cultures of Suillus bovinus were cultivated in inorganic liquid medium with glucose as a carbon source at 25 °C and continuous supply of oxygen by aeration with compressed air in the dark. Exogenous fructose as sole carbon source yielded about 50% less increase in dry weight than glucose. This resulted from different uptake velocities. Sucrose as sole exogenous carbon source yielded no measurable increase in dry weight. In glucose cultures, activities of all glycolytic enzymes were found. Maximum specific activities varied largely (from about 60 [fructose 6-phosphate kinase] to about 20 000 [triosephosphate isomerase] nmoles · mg protein -1 · min -1 ). Apparent K m -values also varied over more than two orders of magnitude (0.035 mᴍ [pyruvate kinase] to 6.16 mᴍ [triosephosphate isomerase]). Fructose 6-phosphate kinase proved to be the fructose 2,6-bisphosphate-regulated type, aldolase the divalent cation-dependent (class II) type and glyceratephosphate mutase the glycerate 2,3-phosphate-independent type of the respective enzymes. Eight of the 10 enzymes exhibited pʜ-optima′ between 7.5-8.0. Triosephosphate isomerase and pyruvate kinase showed highest activities at pʜ 6.5. Regulatory sites within the glycolytic pathway of Suillus bovinus are discussed; fructose 6-phosphate kinase appears to be its main bottle neck.

Gibberellic acid was fed to a homogenate of Mogami-Benibana, a cultivar of dyer’s saffron (Carthamus tinctorius), at a 100 μᴍ level. After incubation for 60 min at 25 or 37 °C, the effect of externally supplied gibberellic acid on enzyme activities and the glucose level were investigated. Gibberellic acid enhances the activity of glucose oxidase by about 52%. It induces also a weak (9%) increase of glucosidase activity. Gibberellic acid increases the glucose content by about 6% during the initial 30-min incubation. The results relate to the red colour appearance in Mogami-Benibana at the later stage of blooming.

In the unicellular green alga Scenedesmus obliquus the level of photoinhibition and the recovery of the cells after reversal to the initial light conditions in relation to the pre-photoadaptation of the culture to low, medium and high light intensity was determined. The changes in the photosynthetic pigment content and in the intracellular polyamine concentration allowed to distinguish between photoadaptation and photoinhibition. In particular, the level of chlorophylls, xanthophylls and carotenoids decreased inversely proportional to the light intensity applied during photoadaptation, whereas their concentrations remained constant during photoinhibition. The violaxanthin/zeaxanthin and the loroxanthin/lutein cycle work only under photoinhibitory conditions, but not under photoadaptive premises. Changes in the level of these carotenoids in relation to the changes in the photosynthetic apparatus during photoadaptation are discussed. In addition, it was found that the intracellular polyamine level increased only under stress conditions, i. e. during photoinhibition, and decreased during recovery of the cells after reversal to the initial light conditions. The increase of the putrescine level during photoinhibition is inversely proportional to the light intensity used for pre-adaptation. This rise of the polyamine level in the cells photoadapted to high light conditions is an additional indication for the finding that photoadaptation and photoinhibition are different phenomena which are clearly distinguishable from each other. Finally, the changes of the chlorophyll, violaxanthin, zeaxanthin, loroxanthin, lutein and polyamine levels under photoadaptation in high light intensity (50 W m -2 ) in relation to the range of photoadaptation in Scenedesmus obliquus are discussed.

CP26, CP29 and three different LHC II subcomplexes have been purified from a carnation photosystem II (PSII) preparation using non-denaturing isoelectric focusing in a vertical polyacrylamide slab gel. The identity of the fractions was established by absorption spectroscopy, SDS-PAGE and immunoblotting. CP26 comprised a single apoprotein of 26.6 kDa and CP29 contained two apoproteins of 28.8 and 28.5 kDa. LHC II subcomplex A consisted of Lhcb1 homotrimers, and subcomplexes B and C consisted of Lhcb1/Lhcb2 and Lhcb1/Lhcb3 heterotrimers, respectively. We discuss the data in relation to the organization of the PS II antenna in vivo.

Stimulatory effect of divalent cations like calcium (Ca 2+ ) and magnesium (Mg2+) was investigated on electron transport activity of divalent cation deficient low-salt suspended (LS) thylakoid preparation from a submerged aquatic angiosperm, Hydrilla verticillata. Both the cations stimulated electron transport activity of LS-suspended thylakoids having an intact water oxidation complex. But in hydroxylamine (NH 2 OH) - or alkaline Tris - washed thylakoid preparations (with the water oxidation enzyme impaired), only Ca 2+ dependent stimulation of electron transport activity was found. The apparent K m of Ca 2+ dependent stimulation of electron flow from H 2 O (endogenous) or from artificial electron donor (exogenous) to dichlorophenol indophenol (acceptor) was found to be identical. Calcium supported stimulation of electron transport activity in NH 2 OH - or Tris - washed thylakoids was electron donor selective, i.e., Ca 2+ ion was only effective in electron flow with diphenylcarbazide but not with NH 2 OH as electron donor to photosystem II. A magnesium effect was observed in thylakoids having an intact water oxidation complex and the ion became unacceptable in NH 2 OH - or Tris - washed thylakoids. Indirect experimental evidences have been presented to suggest that Mg 2+ interacts with the water oxidation complex, while the Ca 2+ interaction is localized betw een Y z and reaction center of photosystem II.

The secondary structure of the member of the AKH/RPCH family has been studied by Molecular Dynamics and Langevin Dynamics methods. Molecular dynamics simulation were performed in vacuum, model aqueous solution and simulated membrane. Langevin dynamics simulation was performed using the friction factor γ equal to 2 ps -1 . Molecular dynamics as well as Langevin Dynamics simulation were conducted at 300 K. All minimum energy conformers have similar backbone structure characterised by the turn consisted out of 3 amino acids, Thr, Pro and Asn 7 . Structures obtained from Molecular Dynamics simulation are characterised by the lack of hydrogen bonds whereas the structure obtained form Langevin Dynamics simulation is stabilised by the web of hydrogen bonds.

The immobilization of catalase was carried out from enzyme solutions in acid (pH < 3.5) and alkaline (pH > 11) medium on two kinds of soot differing by the average size of the particles. Under these conditions the peroxidase-like activity of immobilized catalase in the oxidation of phenol has been studied. The effect of the initial concentration of the substrate on the rate of the process catalysed by catalase immobilized on the soot of finer-grained structure has been studied. The relationships obtained are described by the equation of Michaelis- Menten. The kinetic parameters (the constant of Michaelis - K m , the maximum reaction rate - V max , the rate constant - k and the activation energy - E a ) of the process were calculated. It was found that catalase absorbed on the soot of larger globular particles does not take part in the peroxidase oxidation of phenol.

We have observed that in the absence of hydrogen peroxide the Fe(III)-bleomycin (BLM) complex exhibits high DNA cleavage efficiency, converting supercoiled Form I DNA (pBR322 or Φxl74) to Form II (nicked, relaxed circular); the present study may give an important clue to elucidate the fact that iron-bleomycin mediated double-strand DNA cleavage requires at least one molecule of oxygen (O 2 ) over the amount required to form “activated bleomycin”.

To study the scavenger properties four naphthenic methyl esters were synthesized. The abilities of the compounds to act as radical scavengers were investigated in an experimental epinephrine/adrenochrome model using enzymic and non-enzymic systems. We found that our particular compounds exhibited radical scavenger activity and superoxide-dismutase activity inhibition. Investigated compounds also showed physiological activity. In the auxin test one compound induced elongation of the wheat coleoptile by 35%.

9-hydroxy-10,12-octadecadienoic acid (9-HODE) is generated by lipid peroxidation (LPO) processes in comparison to other marker compounds in at least 10 fold amount. A 10-25 fold increase of this new marker compound in relation to age matched healthy individuals was observed in the low density lipoprotein (LDL) fraction of ten patients suffering from a hemorrhagic shock. The 9-HODE values dropped to normal levels after recovery. Similarily the 9-HODE content in LDL of patients which had to undergo orthopedic surgery - replacement of their arthritic hip joints by endoprosthesis - were investigated. The rather high HODE values dropped also after recovery reflecting obviously the disappearance of inflammatory processes associated with arthritis.

Cholesteryl 3”,4”-dimethoxycinnamate (7) and a new synthesized o-coumaroyl ester of 3β- (2’-hydroxyethoxy)-cholest-5-en (13) exhibited a marked activity against poliovirus type 1 (Mahoney). Compound 7 showed an approximately 20-fold greater selectivity in its antiviral activity than compound 13. These compounds were selected from thirteen steryl esters of cinnamic acid derivatives through an in vitro antiviral screening against viruses belonging to taxonomic groups with causative agents of important human infectious diseases to which chemotherapy is indicated, i.e. Picornaviridae, Orthomyxoviridae, Paramyxoviridae and Herpesviridae.

One of the possible roles of phosphonolipids is that they have a contribution to the protection of cellular integrity and survival of aquatic organisms (mollusca, cnidaria) as these lipids are included at high percentages. The total lipids of the edible mussel Mytilus galloprovincialis (Mollusca, Bivalvia, Mytilidae) were found to constitute 1.27% of fresh tissue. Polar lipid components constitute 61.5% of the total lipids. After separation by Solid Phase Extraction, the polar lipid fraction was separated by two dimensional thin-layer chromatography and the total phosphorus of each component, was determined. The main polar lipids found were: Phosphatidylcholine, 41.6 ± 0.8% (of which 11.3 ± 0.5% was glyceryl ether analog); ceramide aminoethylphosphonate 11.2 ± 0.2% plus 2.8 ± 0.1% another minor species; phosphatidylethanolamine, 26.6 ± 0.5% (of which 12.2 ± 0.3% was glyceryl ether analog). The individual ceramide aminoethylphosphonate species were isolated by preparative thin layer chromatography and the structure of the major one was confirmed by a combination of analytical and chromatographic methods. Saturated fatty acyl groups with 16 carbon atoms were the main components (48.4%) of the major ceramide aminoethylphosphonate species. Diglyceride aminoethyl phosphonates were not found in lipids of M. galloprovincialis.

Protein synthesis in rabbit reticulocyte lysates in the presence of heme is inhibited by 50% by the addition of 4 mᴍ GSSG (oxidized glutathione). The incubation of the rabbit reticulocyte lysate with 4 mᴍ GSSG at 30 °C for 30 min will cause activation of an inhibitor of protein synthesis which could be purified from the lysates through a five-step procedure. The inhibitor results in a 70-80% inhibition after a 1 h incubation. The inhibitor consists of one polypeptide of 23 kDa apparent molecular weight and is 90% pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, in the presence of cAMP (10 mᴍ) or GEF (guanine nucleotide exchange factor) (0.3 μg), protein synthesis in the inhibited reticulocyte lysate will be already recovered.

The main linear epitopes of π-glutathione transferase (π-GST, EC 2.5.1.18), an enzyme related to cancer progression in a restricted number of tumours, were identified by testing in ELISA the reactivities of polyclonal anti-π-GST rabbit sera against a panel of 51 overlapping decapeptides, covering the whole 216-residue sequence of the protein. Several major reactivity peaks were detected, each covering two or three adjacent peptides. The most active fragments were then reconstructed by conventional solid-phase synthesis, linked to Sepharose, and used as affinity ligands for isolating specific anti-π-GST antibody subsets. A second group of antisera was then prepared in rabbits by using as immunogens some of the above described synthetic fragments, linked to a carrier protein, and antipeptide antibodies purified by affinity chromatography. An ELISA test was then performed, using as antigens a panel of peptides and different isoforms of GST, in order to establish whether antibodies isolated from total anti-π-GST sera would display higher reactivity and specificity, as compared to traditional antipeptide antibodies. Binding data clearly confirm that the formers might be indeed better reagents for the detection and possibly quantitation of π-GST.

The presence and regulation by hyperosmotic medium of the ouabain-sensitive (Na + +K + )ATPase of the Malpighian tubule cells of Rhodnius prolixus was investigated. The ouabain-sensitive (Na + +K + )ATPase activity was 5.4 ± 0.5 nmol Pi x mg -1 x min -1 . Vanadate 100 μM completely abolished this ATPase activity. In hyperosmotic medium, obtained by addition of 180 mᴍ mannitol, the (Na + +K + )ATPase activity was inhibited by 60%. When the cell lysates were preincubated in hyperosmotic medium for 30 minutes and the ATPase activity was assayed in isosmotic medium, the (Na + +K + )ATPase activity was not modified. Addition of 50 ng/ml sphingosine, a protein kinase C inhibitor, abolished the inhibition of (Na + +K + )ATPase activity in hyperosmotic medium. Furtherm ore, phorbol ester (TPA), an activator of protein kinase C, mimicked the effect of hyperosmotic shock on (Na + +K + )ATPase activity. The increase in Ca 2+ concentration decreased the (Na + +K + )ATPase activity by 60% in isosmotic medium, with maximal effect obtained in 10 -6 m Ca 2+ . No effect was observed in hyperosmotic medium. The inhibitory effect of Ca 2+ on the (Na + +K + )ATPase was not reversed by sphingosine. These results indicate that the ouabain-sensitive (Na + +K + ) ATPase activity of the Malpighian tubule is regulated by both increasing Ca 2+ concentration and by the osmolality of the medium by different and integrative ways.

Two new saponins were isolated from faba bean ( Vicia faba L.) by column chromatography (Sephadex LH-20 and ODS) and semi-preparative HPLC. Tlieir molecular weights determined by FAB-MS were 980 and 964, respectively. Results of TLC analyse showed that these compounds are similar to soya saponin group B. The presence of separated saponins was confirmed by TLC for seeds of four cultivars of faba bean.

Polyacrylamide gel electrophoresis has been widely used to study DNA fragments containing sequence-dependent curvature. The anomalous electrophoretic behavior of curved DNA fragments on such gels allows their separation from straight fragments of the same length. Here we demonstrate that polyacrylamide gels can be successfully used to resolve DNA fragments modified at a single site by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) from their unmodified counterparts. However, the resolution strongly depends on the voltage gradient, being completely lost when it drops below a certain threshold level. The param eters of the electric field do not affect separation of ‘normal’ DNA fragments of comparable length.

Effects of reabsorption of chlorophyll fluorescence were investigated in measurements on chloroplasts and higher plant leaves. The excitation was perform ed at 650 nm in a typical excitation/detection arrangement; with increasing chlorophyll concentrations, the measured spectral distribution of the chlorophyll fluorescence was significantly distorted, but no effects on the picosecond decay were observed, when this was detected at 685 nm.

N-Acetyl-5-methoxytryptamine (melatonin; MEL) was detected in the testis of non-diapausing pupae and in the testis of post-diapause pharate adults of the cabbage armyworm, Mamestra brassicae, by use of a threedimensional HPLC system with multiple coulometric electrochemical detectors. Intermediates providing evidence of various metabolic pathways were identified, as follows: tryptophan (TRP) → 5-hydroxytryptophan (5-HTP) → 5-hydroxytryptamine (5-HT) → N-methyl-5-hydroxytryptamine (N-MET) ↳5-hydroxyindoleacetic acid (5-HIAA) ↳N-acetyl-5-hydroxytryptamine (N-ACET-5-HT) ↳melatonin (MEL). The possible physiological roles of melatonin in the testis of both diapausing and non-diapausing pupae are discussed.

The secretions of the paracloacal glands of an adult male broad-snouted caiman (Caiman latirostris) were analyzed by GC/MS. 3,7-Dimethyl-6-octen-1-ol (citronellol) and four citronellyl esters, with acid moieties ranging from two to five carbons, were identified. Squalene, β-farnesene, and straight-chain esters also were observed.

A simple method is proposed for the treatm ent of the acquired immunodeficiency syndrome (AIDS) by introducing into the blood stream magnetoliposomes with coupled human immunodeficiency virus (HIV) receptor proteins. After some time lag, needed for binding of HIV and HIV-infected cells to magnetoliposomes, the arterio-venous shunt, as used with dialysis patients should be inserted and the patient’s blood should be passed through multiple tubes filled with stainless steel wool, each of which is surrounded by a coil producing a strong non-homogeneous magnetic field, which may result in a substantial reduction of HIV and HIV-infected cells in the infected body.