Volume 53, Issue 11-12

The three protoilludanes radulone A (1), radulone B (2) and radudiol (3), the illudalane radulactone (4) and the illudane radulol (5) were isolated from the extracts of the culture fluids of the basidiomycete Radulomyces confluens. The structures of the five new compounds were determined by spectroscopic techniques. Radulone A (1) is a potent inhibitor of human and bovine platelet aggregation stimulated by different agonists, inhibiting preferentially the aggregation of human platelets induced by ADP with an IC 50 value of 2 μᴍ . In addition 1 exhibits cytotoxic and antimicrobial activities. The other four compounds exhibited weak antimicrobial and cytotoxic activity.

Biflavonoids are well known to occur in many Gymnospermae. Here we report on their occurrence in epicuticular material of a number of species. Several compounds are charac­terized by detailed NMR studies and FAB-MS. They are identified as amentoflavone, bilobe-tin, podocarpus-flavone A, sciadopitysin, dihydrosciadopitysin and cupressuflavone.

Phosphorylase Phosphatase, Protein Phosphatase, Saccharomyces cerevisiae Aphosphatase, active towards phosphorylase a and phosphorylated proteins casein and histone II-A , was isolated from Saccharomyces cerevisiae 257. The enzyme dephosphorylated glycogen phosphorylase from commercial yeast rendering it inactive. The protein phosphatase activity was not influenced by any metal ions. Phosphorylase phosphatase activity was slightly stimulated by p -nitrophenyl phosphate and inhibited by heparin.

A crude enzyme extract from Dioscorea esculenta var. fasiculata tissue subjected to ion exchange chromatography on DEAE-Sephadex A-50 column. This proced ure resolved the extract into two main protein peaks one of which eluted through the column relatively unbound while the other protein peak which remained bound to the column was eluted with 1.0 ᴍ NaCl. Both protein peaks contained polyphenol oxidase (PPO) and peroxidase (POD) activities. The non-binding protein peak was resolved by gel filtration on Sephadex G-200 into distinct PPO and POD activities and by virtue of their apparent molecular weights of 95.5 Kd and 38.0 Kd for PPO and POD respectively were determined to be the typical enzymes. The PPO activity was completely inhibited invitro by 5 mᴍ polyvinyl pyrrolidone (PVP). The binding protein peak was not resolved by gel filtration. It contained PPO activity which was not inhibited by PVP and a POD activity which was completely inhibited by dithiothreitol (DTT) This ionic protein peak contained 60% of total POD in the tissue, has an apparent molecular weight of 56 Kd and is suggested to be a strongly anionic peroxidase which also exhibits polyphenol oxidase activity.

Phototrophic continuous and batch cultures of Rhodobacter capsulatus were employed to identify the C/N ratio above which nitrogenase is de-repressed. The cultures were grown with limiting amounts of ammonium as source of bound nitrogen and with ʟ-lactate or ʟ-malate as sources of carbon and reducing equivalents. De-repression of nitrogenase was determined on the basis of the occurrence of dinitrogen fixation, acetylene reduction and nifH promoter activities as well as on the basis of hydrogen evolution and nitrogenase poly­ peptides. In continuous culture, cells started to fix dinitrogen, to reduce acetylene, to activate the nifH promoter and to form nitrogenase polypeptides, when consuming lactate per ammonium at a C/N ratio of about 6 (this ratio represents the number of C and N atoms consumed). With malate as carbon source all of the activities becam e detectable above a C/N ratio of about 8. Essentially the same C/N ratios were determined with batch cultures for the occurrence of N -limitation of growth and hydrogen evolution. The experimentally deter­mined C/N ratios for nitrogenase de-repression essentially agreed with C/N ratio of 5.8 and 7.8 calculated for the assimilation of ammonium and either lactate or malate, into biomass of an elemental com position of CH 1.83 N 0.183 O 0.5 -This means that the occurrence of N -limitation and nitrogenase de-repression is defined by a threshold C/N ratio required for biomass production. As experimentally and theoretically shown, this ratio depends on the reduction state of the carbon source. It is concluded that the C/N ratio of nutrient consumption represents an intracellular signal which is directly translated into nitrogenase de-repression.

The vegetable sponge of Luffa cylindrica was studied as a matrix for the immobilization of Aspergillus niger 26. p roducer of polymethylgalacturonase (PMG). Entrapped spores could grow and multiply within the lattice of the sponge. The influence of loofa sponge inoculum content, initial spore inoculum content, and du ration of the growth cycle on the enzyme activity and mycelium growth was studied. The best yield of PMG was reach ed with 1 piece of loofa sponge (approx. 0.10 g dry weight), 10 9 spores per g carrier and 48 h duration of one cycle. Data obtained during long-term semicontinuous cultivation showed that production capacity increased significantly and the production period was extended more than 10 times compared with the free cell culture.

The subcellular distribution of glutathione S-transferase (GST) activity extracted from shoots of 3-day-old etiolated seedlings of maize (Zea mays L., Northrup-K ing 9283 hybrid) and the induction of soluble and m em brane-bound G ST activity by the safener benoxacor, the herbicide metolachlor and their com bination (CGA -180937) were investigated. GST activity extracted from maize shoots was detected in both cytosolic and microsomal fractions and utilized l-chloro-2,4-dinitrobenzene (CDNB ), metolachlor, and trans-cinnamic acid (CA) as substrates. Soluble GST activity extracted from maize shoots was greater than m icrosom al with CDNB or metolachlor as substrate. Membrane-bound GT activity was greater than soluble with cinnamic acid as substrate. W ashing the microsomal preparations from maize shoots with Triton X-100 increased GST (C A ) activity. Pretreatment with the safener benoxacor or a form ulated combination of the herbicide metolachlor with benoxacor induced soluble GST(CDNB ), GST(metolachlor) and GST (C A ) activities in m aize shoots. Benoxacor and CGA-180937 induced also membrane-bound GST (CDNB ) and GST(CA ) activities in maize shoots, but did not affect membrane-bound GST (metolachlor) activity. These results confirm that maize contains multiple GST isozymes that differ in their subsrate specificity and inducibility by safeners or other chemicals.

Various bacterial and plastidic plant terpenoids are synthesized via the non-mevalonate1-deoxy-ᴅ-xylulose-5-phosphate (DOXP) pathway. The antibiotic and herbicidal compound fosmidomycin is known to inhibit growth of several bacteria and plants, but so far its mode of action was unknown. Here we present data which demonstrate that the DOXP pathway of isoprenoid biosynthesis is efficiently blocked by fosmidomycin. The results point to the DOXP reductoisom erase as the probable target enzyme of fosmidomycin.

Nickel(II) salts and their coordination com pounds with ethyl 5-m ethylim idazole-4-carboxylate (emizco), [Ni(emizco) 2 Cl 2 ], [Ni(emizco) 2 Br 2 ], [Ni(emizco) 2 (H 2 O) 2 ] (NO 3 ) 2 H 2 O · Ni(NO 3 ) 2 , inhibit photosynthetic electron flow (basal, phosphorylating and uncoupled) and ATP-synthesis, therefore behave as Hill reaction inhibitors. Coordination compounds are more potent inhibitors than the salts. It was found that the target for NiCl 2 ; NiBr 2 and Ni(NO 3 ) 2 is at the b 6 f level. On the other hand, the complexes [Ni(Emizco) 2 Cl 2 ], [Ni(Emizco) 2 Br 2 ] and [Ni(emizco) 2 (H 2 O ) 2 ] (NO 3 ) 2 H 2 O binding sites are located at Q B (D1)-protein and b 6 f level. Therefore, they have a common inhibition site located at b 6 f avoiding the PQH 2 oxidation. The Q B inhibition site was corroborated by variable chlorophyll a fluorescence yield [V(j)]. The emizco ligand has no activity on photosynthetic electron flow.

Herbicidal chloroacetamides cause a very sensitive inhibition of fatty acid incorporation into an insoluble cell wall fraction of Scenedesmus acutus. The molecular basis was investigated in more detail. After incubation of the algae with [ 14 C]oleic acid and saponification, the remaining pellet was solubilized and fractionated consecutively with chloroform / methanol, phosphate buffer, amylase, pronase, and finally with dioxane/HCl. By acid hydrolysis in dioxane a part of the cell wall residue was solubilized showing inhibition of exogenously applied oleic acid and other labelled precursors such as stearic acid, palmitic acid, and acetate. After extraction of this dioxane-soluble subfraction with hexane, HPLC could separate labelled metabolites less polar than oleic acid. T heir formation was completely inhibited by chloroacetam ides, e.g. 1 μᴍ metazachlor. This effect was also observed with the herbicidally active 5-enantiomer of metolachlor while the inactive R-enantiomer had no influence. These strongly inhibited metabolites could be characterized by radio-HPLC /MS as very long chain fatty acids (VLCFAs) with a carbon chain between 20 and 26. Incubating am etazachlor-resistant cell line of S. acutus (Mz-1) with [ 14 C]oleic acid, V LCFA s could not be detected in the dioxane/ HCl-subfraction. Furthermore, comparing the presence of endogenous fatty acids in wildtype and mutant Mz-1 the VLCFA content of the mutant is very low, while the content of long chain fatty acids (C16 -18) is increased, particularly oleic acid. Obviously, the phytotoxicity of chloroacetam ides in S. acutus is due to inhibition of VLCFA synthesis. The resistance of the mutant to metazachlor has a bearing on the higher amount of long chain fatty acids replacing the missing VLCFAs in essential membranes or cell wall components.

In short-term -experiments [ 14 C]-labelled malonic acid, stearic acid and acetate have been incorporated into leaf fatty acids of seedlings of Cucumis sativus, Hordeum vulgare and Zea mays. The pattern of labelled fatty acids changed markedly by treatment with the chloroacetamide herbicides metazachlor, metolachlor or butachlor. During a 2-h incubation time, 1 μᴍ chloroacetamide specifically inhibited up to 100% the formation of the saturated very long chain fatty acids (V LCFAs) with a carbon number of 20, 22 and 24. In cucumber and barley a 50% inhibition of VLCFA formation is achieved with 10 to 100 nM metazachlor representing the most sensitive effect of inhibitors on fatty acid elongation reported as yet. Sensitivity of fatty acid elongation depends on the amide structure present in the com pound and on its stereochemistry. Inhibition of oleic acid incorporation correlates with growth inhibition by chloroacetam ides of the intact cell (comp. Pestic. Sei. 52, 381-387, 1998). The present study extends this correlation to inhibition of VLCFA synthesis in higher plants. Obviously the primary mode of action of chloroacetam ides and related herbicidal substances is involved in the enzymic four-step fatty acid elongation system.

Soybean β-1,3-endoglucanase represents a model system for studies on early plant re­sponses to infection by fungal pathogens, and it has been implicated in the release of elicitors from fungal cell walls. In the present study, potato plants were transformed with the soybean β-1,3-endoglucanase cDNA via Agrobacterium delivery system. The transfer of the gene into potato genome was confirmed by (i) PCR amplification, (ii) Northern blot analyses, and (Hi) an increase in the activity of β-1,3-endoglucanase in transgenic plants. The transformation resulted in an increased resistance of selected transgenic plants to infection by Phytophthora infestans, an important pathogen.

Two proteins with apparent molecular masses of 17 and 24 kD that are synthesized in a circadian manner in the phytoflagellate Euglena gracilis, were recognized as proteins belong­ing to the family of light-harvesting-chlorophyll-proteins (LHCPs) of class I (17 kD) and of class II (24 kD). Identification was achieved by N-terminal sequencing of the proteins isolated from two-dimensional polyacrylamide gels and by detection with an anti-LHCP II se­rum. While it was found that the total amount of LHCPs remains almost constant, when Euglena is grown under diurnal conditions (12 h light and 12 h dark), we could show that the amount of newly synthesized 17 and 24 kD proteins varies about 20-fold with a maximum of synthesis in the light phase. In contrast, the analysis of the mRNA levels at different times revealed only minor differences in the stationary concentration of the LHCP specific mRNA, indicating that the control of LHCP synthesis is at the translational level. Principally, the same finding was obtained using inhibitors of transcription. Thus, it is concluded that the expression of LHCPs in Euglena gracilis in contrast to that of higher plants is primarily regulated at the translational level.

The results of theoretical geometry prediction of formamidopyrimidine(fapy)-adenine and fapy-guanine tautomers are presented. Among 54 potential tautomeric structures of fapy-adenine the most stable structure corresponds to the diamino-keto isomer. The solvent effect has insignificant influence on the fapy-adenine tautomers succession. The fapy-guanine has 172 potential isomers. There are three most stable tautomers of this guanine derivative, which may exchange the order depending on the polarity of the environment. In vapour the most probable is the 4-enol-6-keto-diamino tautomer, while in water environment the 4,6-diketo-diamino isomer is dominant. A more polar solvent stabilises more polar fapy-guanine tauto­mers. The geometric parameters and point-atomic charges corresponding to most probable tau­tomers are also supplied.

The methanol extract of Brazilian propolis was fractionated by HPLC, based on human hepatocellular carcinoma (HuH 13) cell cytotoxicity assay. A new benzo-y-pyran derivative (PM-3) with a molecular formula of C 19 H 22 O 3 (MW: 298.38) was isolated. The structure of this colorless compound was determined as 3-[2-dimethyl-8-(3-methyl-2-butenyl) benzopy-ran]-6 -propenoic acid. This compound was chemically synthesized by cyclisation of artepillin C (3-[4-hydroxy-3,5-bis (3-methyl-2-butenyl) phenyl]-2-propenoic acid).

extract of propolis (EEP) has antibacterial, antiviral, antiprotozoal and antifungal properties, in addition to many biological effects. Our laboratory has demonstrated a syner­gistic effect of EEP and antibiotics on the growth of Staphylococcus aureus, and suggested that the bactericidal effect of EEP was expressed mainly on virulent mycobacteria rather than on non-virulent (attenuated) ones. The present study was designed to reconfirm the latter finding, by subjecting 17 different mycobacteria strains to EEP, and evaluating whether there is a correlation between the virulence of the mycobacteria strains studied and their susceptibility to EEP. Our findings demonstrate that while the four non-virulent strains studied are not susceptible to EEP, out of the 13 virulent strains studied seven are susceptible and six are resistant to it. These results suggest that while there is no full correlation between virulence of the mycobacteria tested and their susceptibility to EEP, the few strains that were resistant to EE P were non-virulent.

The purpose of this investigation was to determine the antimicrobial and healing potential of propolis on direct dental pulp exposures. This study used 25 adult male rats. Pulp expo­sures were performed and animals were allocated to propolis and calcium hydroxide (Ca(OH) 2 groups. Animals were killed on days 5, 7, 10, and 14. The teeth were routinely processed for histological evaluation. Non-parametric tests were employed to analyze the data. No significant differences were found between study groups on the wound healing of the dental pulp. Both substances were com parable in exhibiting normal reorganization of the pulp and no increased vascularity, and were equally efficacious in maintaining a low inflammatory and microbial cell population as well as in stimulating the formation of reparative dentin.

The presence of (Na + +K + )ATPase activity in CL14 clone and NIH NTY strain of Trypano­soma cruzi epimastigotes is demonstrated. A Na + plus K + stimulated ATPase activity is found in both strains. The optimal Na + /K + ratio is 5:1 and 9:1 in CL14 clone and NIH NTY strain, respectively. In both strains, vanadate completely inhibits the ouabain-sensitive ATPase activ­ity indicating that it belongs to the P-type (E 1 /E 2 ) family of ion-transporting ATPases. The I 50 for vanadate is 0.66 ± 0.04 and 0.04 ± 0.02 μᴍ in CL14 clone and NIH NTY strain, respectively. These data indicate that both strains of T. cruzi epimastigotes express the oua­ bain-and vanadate-sensitive (Na + +K + )ATPase activity. On the other hand, the discrepancy between the parameters analyzed for the inhibitors suggests that they express different iso­ forms of this enzyme.

The effect of different L-phenylalanine (Phe) concentrations (0.24-12.1 mᴍ) , on acetylcho­linesterase (AChE) and Na + ,K + -ATPase activities of diaphragm homogenates from 21-day old rats and pure enzymes, was investigated at 37 °C. AChE and N a + ,K + -ATPase activities were determined after preincubation with Phe. AChE activity in diaphragm homogenate or in pure eel E. electricus enzyme showed a decrease, which reached a maximum of 18% with Phe concentrations of 0.9-12.1 mᴍ. However lower Phe concentrations (0.24 mᴍ) increased the enzyme activity (by approximately 22%), only in the diaphragm homogenate. Dia­phragm-associated Na + ,K + -ATPase activity showed a progressive and concentration-depen­ dent decrease, by about 30-35% in the presence of high Phe concentrations. Pure enzyme activity (from porcine cerebral cortex) was not affected by high Phe concentrations (>0.48 m M) , while it was increased by low concentrations. The above results suggest; a) A direct inactivating effect of high Phe concentrations on AChE and an indirect activating effect induced by low concentrations, b) A direct activating effect of low Phe concentrations and an indirect inactivating effect of high ones on Na + ,K + -ATPase. c) The combination of high Phe concentrations effects on AChE and Na + ,K + -ATPase could influence the levels of the diaphragm synaptic ACh.

The new indicator for lipid peroxidation (LPO) processes -9-hydroxy-10,12-octadecadie-noic acid (9-HODE) -was used to investigate, whether LPO processes are increased in destructed bone material of patients suffering from rheum atoid arthritis (RA) in comparison to surrounded non destructed bone material. The HODE content in destructed bones ex­ceeded that of non destructed ones of the same patient for a factor of about 3. In addition similar increases in leukotoxines and epoxy oleic acid in the destructed bone material were observed, indicating an increase of LPO processes in affected bone parts of pa­tients.

Rheumatic diseases are accompanied by a progradient diminution of the cartilage layer. Unfortunately, degradation mechanisms (role of different enzymes and reactive oxygen spe­cies) are not yet understood. Since nuclear magnetic resonance (NMR) spectroscopy was often used for the investigation of synovial fluids, the aim of the present work was to detect cartilage degradation products upon proteolytic digest of cartilage. Cartilage samples were incubated at 37 °C with phosphate buffer in the absence or pres­ence of different proteases (collagenase, trypsin and papain). Supernatants were subsequently assayed towards their content of carbohydrate and protein degradation products by NMR ( 1 H-and 13 C-) and MALDI-TOF mass spectrometry. Intense resonances of relatively mobile N-acetyl side chains (ca. 2.01 ppm) of polysaccha­ rides of cartilage were only detectable on digestion with papain. The appearance of these resonances indicates intense degradation of the core protein of proteoglycan aggregate of cartilage, whereby polysaccharides are released. Additionally, broad resonances at 0.85 ppm arising from collagen degradation products were observed upon the action of all applied proteases. However, glycine as a m arker of exhaustive collagen degradation was only ob­ served, if cartilage was digested by collagenase. Using more vigorous incubation conditions, additionally high-abundant amino acids of collagen (proline, hydroxyproline) could be de­ tected in the 13 C-NMR-and the MALDI spectra. The observed differences are correlated with the different selectivities of the applied enzymes. It is concluded that NMR allows the detection of characteristic protein and polysaccharide degradation products. The observed differences may be of some relevance for the diagnosis of rheumatic diseases.

Pederin analyses of single Paederus riparius specimens reared from the egg and kept for prolonged periods of time show that only the females are able to biosynthesize the substance. Preimaginal stages efficiently store pederin transferred by the females into their eggs and the males’ pederin content decreases slowly over time. Only males with access to eggs con­ taining the substance moderately increase their pederin load. The females begin to accumu­ late the toxin a few weeks after imaginal eclosion and build up reserves for the egg laying period within 60 days. This is discussed with regard to the life cycle of P. riparius. During reproduction, when the females loose pederin due to maternal transfer, they must replenish their pederin reserves. This biosynthesis is not reflected in the amount found per individual.

Two new headspace methods for the analysis of iso­prene emission from detached plant leaves are described. The first method is based on the UV-absorption of isoprene emitted by leaves inside quartz cuvettes and can be used for the quantitative spectrophotometric determination of isoprene production. The second tech­nique is a micro-extraction method of isoprene from the cuvette air for GC analysis, and is very suitable for the determination of isotope-labeled isoprene by GC-MS.

Six hydroxycinnamic acids : caffeic acid, chlorogenic acid, 3,5-O-dicaffeoylquinic acid 2-O-caffeoyltartaric acid (caftaric acid) and 2,3-O-dicaffeoyltartaric acid (chicoric acid) have been isolated from Lapsana communis L. subsp. communis aerial parts. Among flavonoids, only isoquercitrin, luteolin and luteolin-7-O-β-glucuronide were identified. Except for chlorogenic acid, these com­ pounds represent the first report in Lapsana communis. Chicoric acid is the major phenylpropanoic constituent in this plant.

The cytostatic activity of seven phenolic acids: coumaric, caffeic, ferulic, gentisic, protocatechuic, syringic and isovanillic acid previously isolated from Scrophularia frutescens has been evaluated against Hep -2 cells (de­rived from a human epidermoid carcinoma of the lar­ynx) and McCoy cells (derived from the synovial fluid in the knee joint of a patient suffering from degenerative arthritis). The compounds belonging to the cinnamic group present the highest activity.

The fine structure and water permeability of hypoder­mis isolated from roots of Clivia miniata have been studied. The hypodermis is composed of five layers of cells arranged in radial rows. The cell walls of these lay­ers consist of primary and tertiary walls and suberized secondary walls which are lamellated. Water perm eabil­ity of the isolates was low, around the value of 10 -9 m s -1 . This value was found independent of the pH solu­tion and of the ionic exchange capacity of the isolates. Suberin extraction increased water permeability one or­ der of magnitude.