In vitro evidence that the anti-inflammatory effect of synthetic cinnamate-derived dienes is directly linked to a macrophage repolarization.

The inflammatory process is a mammalian physiological reaction against infectious agents or injuries. Among the cells involved, the macrophages have a highlighted role during this process. Depending on the inflammatory context, they can polarize into pro- or anti-inflammatory profiles (M1 and M2). In this context, compounds derived from cinnamic acid have demonstrated strong evidence of anti-inflammatory activity; however, the mechanism responsible for this effect remains unclear. In this study, we investigated the anti-inflammatory activity of five cinnamate-derived dienes of synthetic origin. The compounds that did not demonstrate significant cytotoxicity were tested to assess anti-inflammatory activity (NOx ) in RAW 264.7 cells stimulated with LPS. Then, the selected compound (diene 1) was evaluated as to its ability to inhibit the secretion of pro-inflammatory cytokines (IL-1ß, TNF-α, INF-γ, MCP-1, and IL-6) and increase the production of anti-inflammatory cytokines (IL-13, IL-4, and IL-10). Finally, diene 1 was able to reduce the expression of TLR4 and increase the phagocytic activity of the macrophages. Gathering these results together, we conclude that diene 1 showed an important anti-inflammatory effect, and this effect is linked to its immunomodulatory characteristic. Since the M1 markers were reduced at the same time, M2 markers were increased by the treatment of the macrophages with diene 1.

Meaningful Words in Crowd Noise: Searching for Volatiles Relevant to Carpenter Bees among the Diverse Scent Blends of Bee Flowers.

Olfactory cues constitute one of the most important plant-pollinator communication channels. Specific chemical components can be associated with specific pollinator functional groups due to pollinator-mediated selection on flower volatile (FV) emission. Here, we used multivariate analyses of FV data to detect an association between FVs and the worldwide distributed pollinator group of the carpenter bees (Xylocopa spp.). We compiled FVs of 29 plant species: 9 pollinated by carpenter bees, 20 pollinated by other bee pollinator functional groups. We tested whether FV emission differed between these groups. To rule out any phylogenetic bias in our dataset, we tested FV emission for phylogenetic signal. Finally, using field assays, we tested the attractive function of two FVs found to be associated with carpenter bees. We found no significant multivariate difference between the two plant groups FVs. However, seven FVs (five apocarotenoid terpenoids, one long-chain alkane and one benzenoid) were significantly associated with carpenter bee pollination, thus being "predictor" compounds of pollination by this pollinator functional group. From those, ß-ionone and (E)-methyl cinnamate presented the highest indicator values and had their behavioural function assessed in field assays. Phylogenetic signal for FVs emission was weak, suggesting that their emission could result from pollinator-mediated selection. In field assays, the apocarotenoid ß-ionone attracted carpenter bees, but also bees from other functional groups. The benzenoid (E)-methyl cinnamate did not attract significant numbers of pollinators. Thus, ß-ionone functions as a non-specific bee attractant, while apocarotenoid FVs emerge as consistent indicators of pollination by large food-foraging bees among bee-pollinated flowers.

Cinnamoyl-N-Acylhydrazone-Donepezil Hybrids: Synthesis and Evaluation of Novel Multifunctional Ligands Against Neurodegenerative Diseases.

A new series of ten multifunctional Cinnamoyl-N-acylhydrazone-donepezil hybrids was synthesized and evaluated as multifunctional ligands against neurodegenerative diseases. The molecular hybridization approach was based on the combination of 1-benzyl-4-piperidine fragment from the anti-Alzheimer AChE inhibitor donepezil (1) and the cinnamoyl subunit from curcumin (2), a natural product with remarkable antioxidant, neuroprotective and anti-inflammatory properties, using a N-acylhydrazone fragment as a spacer subunit. Compounds 4a and 4d showed moderate inhibitory activity towards AChE with IC50 values of 13.04 and 9.1 µM, respectively. In addition, compound 4a and 4d showed a similar predicted binding mode to that observed for donepezil in the molecular docking studies. On the other hand, compounds 4a and 4c exhibited significant radical scavenging activity, showing the best effects on the DPPH test and also exhibited a significant protective neuronal cell viability exposed to t-BuOOH and against 6-OHDA insult to prevent the oxidative stress in Parkinson's disease. Similarly, compound 4c was capable to prevent the ROS formation, with indirect antioxidant activity increasing intracellular GSH levels and the ability to counteract the neurotoxicity induced by both OAß1-42 and 3-NP. In addition, ADMET in silico prediction indicated that both compounds 4a and 4c did not show relevant toxic effects. Due to their above-mentioned biological properties, compounds 4a and 4c could be explored as lead compounds in search of more effective and low toxic small molecules with multiple neuroprotective effects for neurodegenerative diseases.

Metabolomic approach for characterization of phenolic compounds in different wheat genotypes during grain development.

The phenolic-profiling of seven different wheat (Triticum aestivum) genotypes was investigated for the first time during different stages of grain development (milky, softy, physiological maturity and mature). Free and bound phenolic compounds were extracted separately and analyzed by UPLC-QTOF-MSE. Total phenolic content significantly decreased, up to 50% depending on the genotype, towards the maturation of grain. The highest content (free and bound) was observed in the most immature grains, while the lowest level was found in mature grains (408.0 and 165.0 GAE mg/100 g, respectively). Globally, 237 phenolic compounds were identified, divided into 5 classes: flavonoids (85), phenolic acids (77), other polyphenols (51), lignans (16) and stilbenes (8). UPLC-MS results showed a progressively decrease of the number of phenolic identification (ID) all along grain development, milky (213), softy (192), physiological maturity (169) and mature (144). The proportion bound to free phenolic progressively increased, reaching the maximum at physiological maturity, indicating a possible enzymatic reactions and complexation during grain growth. Ferulic acid, diphyllin, 4-hydroxybenzoic acid, ferulic acid isomer, apigenin 7-O-apiosyl-glucoside isomer and myricetin isomer were the most abundant compounds. Chemometric tools showed a clear separation between immature and mature grain for all genotypes. Phenolic profile varied significantly among genotypes, this result can help the selection of varieties towards a higher retention of bioactive compounds. Noteworthy, immature wheat grains can be considered a rich source of phenolic compounds and as an attractive ingredient to incorporate to functional foods.

Cinnamic acids derived compounds with antileishmanial activity target Leishmania amazonensis arginase.

This study describes the activity of five natural hydroxycinnamic acids and derived compound: caffeic (1), rosmarinic (2), chlorogenic (3), and cryptochlorogenic (4), acids and isoverbascoside (5). All compounds inhibited Leishmania amazonensis arginase with IC50 -in range of 1.5-11 µM. Compounds 2 and 5 also showed activity against promastigotes of L. amazonensis with IC50  = 61 (28-133) µM and IC50  = 14 (9-24) µM, respectively. Further computational studies applying molecular docking simulations were performed on the competitive inhibitors to gain insight into the molecular basis for arginase inhibition and could be exploited to the development of new antileishmanials drug targeting parasite arginase.

Volatile phenols are produced by strains of Dekkera bruxellensis under Brazilian fuel ethanol industry-like conditions.

Dekkera bruxellensis is a spoilage yeast in wine and fuel ethanol fermentations able to produce volatile phenols from hydroxycinnamic acids by the action of the enzymes cinnamate decarboxylase (CD) and vinyphenol reductase (VR) in wine. However, there is no information about this ability in the bioethanol industry. This work evaluated CD and VR activities and 4-ethylphenol production from p-coumaric acid by three strains of D. bruxellensis and PE-2, an industrial Saccharomyces cerevisiae strain. Single and multiple-cycle batch fermentations in molasses and sugarcane juice were carried out. Dekkera bruxellensis strains showed similar CD activity but differences in VR activity. No production of 4-ethylphenol by S. cerevisiae in any fermentation system or media was observed. The concentrations of 4-ethylphenol peaked during active growth of D. bruxellensis in single-cycle fermentation but they were lower than in multiple-cycle fermentation. Higher concentrations were observed in molasses with molar conversion (p-coumaric acid to 4-ethylphenol) ranging from 45% to 85%. As the first report on 4-ethylphenol production in sugarcane musts by D. bruxellensis in industry-like conditions, it opens up a new avenue to investigate its effect on the viability and fermentative capacity of S. cerevisiae as well as to understand the interaction between the yeasts in the bioethanol industry.

Processing 'Ataulfo' Mango into Juice Preserves the Bioavailability and Antioxidant Capacity of Its Phenolic Compounds.

The health-promoting effects of phenolic compounds depend on their bioaccessibility from the food matrix and their consequent bioavailability. We carried out a randomized crossover pilot clinical trial to evaluate the matrix effect (raw flesh and juice) of 'Ataulfo' mango on the bioavailability of its phenolic compounds. Twelve healthy male subjects consumed a dose of mango flesh or juice. Blood was collected for six hours after consumption, and urine for 24 h. Plasma and urine phenolics were analyzed by electrochemical detection coupled to high performance liquid chromatography (HPLC-ECD). Five compounds were identified and quantified in plasma. Six phenolic compounds, plus a microbial metabolite (pyrogallol) were quantified in urine, suggesting colonic metabolism. The maximum plasma concentration (Cmax) occurred 2-4 h after consumption; excretion rates were maximum at 8-24 h. Mango flesh contributed to greater protocatechuic acid absorption (49%), mango juice contributed to higher chlorogenic acid absorption (62%). Our data suggests that the bioavailability and antioxidant capacity of mango phenolics is preserved, and may be increased when the flesh is processed into juice.

Immobilized tannase treatment alters polyphenolic composition in teas and their potential anti-obesity and hypoglycemic activities in vitro.

The aim of this work was to assess the effect of immobilized-tannase treatment on black, green, white and mate tea components and on their bioactivities relevant to obesity. Tannase treatment caused predictable changes in polyphenol composition with substantial reduction in galloylated catechins in green, white and black tea. Mate tea, which is rich in chlorogenic acids, was much less affected by tannase treatment although some degradation of caffeoyl quinic acid derivatives was noted. The original tea samples were effective in inhibiting digestive enzymes in vitro. They inhibited amylase activity, some with IC50 values ∼70 µg mL(-1), but were much less effective against α-glucosidase. They also inhibited lipase activity in vitro and caused dose-dependent reductions in lipid accumulation in cultured adipocytes. The bio-transformed tea samples generally matched the effectiveness of the original samples but in some cases they were markedly improved. In particular, tannase treatment reduced the IC50 value for amylase inhibition for green tea and white tea by 15- and 6-fold respectively. In addition, the bio-transformed samples were more effective than the original samples in preventing lipid accumulation in adipocytes. These in vitro studies indicate that bio-transformed tea polyphenols could assist in the management of obesity through improvement in energy uptake and lipid metabolism and also indicate that biotechnological modification of natural food molecules can improve the benefits of a common beverage such as tea.

Combining in vitro and in silico approaches to evaluate the multifunctional profile of rosmarinic acid from Blechnum brasiliense on targets related to neurodegeneration.

Natural products are important sources of chemical diversity leading to unique scaffolds that can be exploited in the discovery of new drug candidates or chemical probes. In this context, chemical and biological investigation of ferns and lycophytes occurring in Brazil is an approach adopted by our research group aiming at discovering bioactive molecules acting on neurodegeneration targets. In the present study, rosmarinic acid (RA) isolated from Blechnum brasiliense showed an in vitro multifunctional profile characterized by antioxidant effects, and monoamine oxidases (MAO-A and MAO-B) and catechol-O-methyl transferase (COMT) inhibition. RA showed antioxidant effects against hydroxyl (HO(•)) and nitric oxide (NO) radicals (IC50 of 29.4 and 140 µM, respectively), and inhibition of lipid peroxidation (IC50 of 19.6 µM). In addition, RA inhibited MAO-A, MAO-B and COMT enzymes with IC50 values of 50.1, 184.6 and 26.7 µM, respectively. The MAO-A modulation showed a non-time-dependent profile, suggesting a reversible mechanism of inhibition. Structural insights on RA interactions with MAO-A and COMT were investigated by molecular docking. Finally, RA (up to 5 mM) demonstrated no cytotoxicity on polymorphonuclear rat cells. Taken together, our results suggest that RA may be exploited as a template for the development of new antioxidant molecules possessing additional MAO and COMT inhibition effects to be further investigated on in vitro and in vivo models of neurodegenerative diseases.

Reduction of L-phenylalanine in protein hydrolysates using L-phenylalanine ammonia-lyase from Rhodosporidium toruloides.

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.25) from Rhodosporidium toruloides was utilized to remove L-phenylalanine (L-Phe) from different commercial protein hydrolysates. A casein acid hydrolysate (CAH, L-Phe ~2.28 %) was employed as a model substrate. t-Cinnamic acid resulting from deamination of L-Phe was extracted, analyzed at λ = 290 nm, and used for PAL activity determination. Optimum reaction conditions, optimized using successive Doehlert design, were 35 mg mL(-1) of CAH and 800 mU mL(-1) of PAL, while temperature and pH were 42 °C and 8.7, respectively. Reaction kinetics of PAL with CAH was determined under optimized conditions. Then, removal of L-Phe from CAH was tested. Results showed that more than 92 % of initial L-Phe was eliminated. Similar results were obtained with other protein hydrolysates. These findings demonstrate that PAL is a useful biocatalyst for L-Phe removal from protein hydrolysates, which can be evaluated as potential ingredients in foodstuffs for PKU patients.

Enhanced lignin monomer production caused by cinnamic Acid and its hydroxylated derivatives inhibits soybean root growth.

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.

Molecular and physiological comparison of spoilage wine yeasts.

AIMS: Dekkera bruxellensis and Pichia guilliermondii are contaminating yeasts in wine due to the production of phenolic aromas. Although the degradation pathway of cinnamic acids, precursors of these phenolic compounds has been described in D. bruxellensis, no such pathway has been described in P. guilliermondii. METHODS AND RESULTS: A molecular and physiological characterization of 14 D. bruxellensis and 15 P. guilliermondii phenol-producing strains was carried out. Both p-coumarate decarboxylase (CD) and vinyl reductase (VR) activities, responsible for the production of volatile phenols, were quantified and the production of 4-vinylphenol and 4-ethylphenol were measured. All D. bruxellensis and some P. guilliermondii strains showed the two enzymatic activities, whilst 11 of the 15 strains of this latter species showed only CD activity and did not produce 4-EP in the assay conditions. Furthermore, PCR products obtained with degenerated primers showed a low homology with the sequence of the gene for a phenyl acrylic acid decarboxylase activity described in Saccharomyces cerevisiae. CONCLUSIONS: D. bruxellensis and P. guilliermondii may share a similar metabolic pathway for the degradation of cinnamic acids. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work that analyses the CD and VR activities in P. guilliermondii, and the results suggest that within this species, there are differences in the metabolization of cinnamic acids.

Nitrate reductase- and nitric oxide-dependent activation of sinapoylglucose:malate sinapoyltransferase in leaves of Arabidopsis thaliana.

Nitrate reductase (NR) activity is necessary for the synthesis of nitric oxide (NO), a key signaling molecule in plants. Here, we investigated the effect of NR deficiency on NO production and phenylpropanoid metabolism of Arabidopsis thaliana leaves. HPLC-mass spectrometry analysis showed that the NR double mutant (nia1 nia2) is deficient in the synthesis of sinapoylmalate (SM), the main phenylpropanoid end-product in wild-type leaves, resulting in accumulation of its precursor sinapoylglucose (SG). While real-time PCR analysis revealed no significant difference at the transcript level, sinapoylglucose:malate sinapoyltransferase (SMT) activity in leaf extracts was reduced in the mutant compared with the wild type. The low levels of SM in nia1 nia2 leaves do not result from the deficient nitrogen incorporation into amino acids, since the recovery of the amino acid content of nia1 nia2 by irrigating the plants with glutamine did not change the metabolic profile of this mutant. In contrast, an increased supply of nitrate stimulated NR activity and NO production, and enhanced SM and decreased SG levels in both genotypes. Nevertheless, sinapic acid esters in nia1 nia2 were not recovered when compared with those detected in the leaves of the wild-type plant. Mutant plants grown in medium supplemented with malate and an NO donor recovered SM to the levels of wild-type leaves. Overall, the results suggest that SMT activity is dependent on the NR-dependent steady-state levels of NO during plant development.

Structural and functional studies of a bothropic myotoxin complexed to rosmarinic acid: new insights into Lys49-PLA2 inhibition.

Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy.

Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with rosmarinic acid.

PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A(2) from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8 A resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P2(1)2(1)2(1), indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A(2) structures belong to space group P3(1)21, while the crystals of complexed structures belong to space groups P2(1) or P2(1)2(1)2(1).

Metabolism of the mesoionic compound (MI-D) by mouse liver microsome, detection of its metabolite in vivo, and acute toxicity in mice.

The mesoionic derivative 4-phenyl-5-[4-nitrocinnamoyl]-1,3,4-thiadiazolyl-2-phenylamine chloride (MI-D) has antitumoral and anti-inflammatory effects. In this study, we present aspects of its metabolism and toxicity in mice. MI-D was metabolized in vitro by liver microsome, generating a main product with a much shorter retention time than MI-D in high-performance liquid chromatography (HPLC) analysis but with a spectrum similar to that of the original molecule. Mass spectrometry with electrospray ionization in positive mode analysis of the purified compound by HPLC indicated that the product of metabolism has four additional hydroxyl groups (m/z = 465) compared with MI-D (m/z = 401). The HPLC analyses of plasma and urine samples from mice treated with MI-D showed the presence of the metabolite product. The kinetic parameters K(m) (19.5 +/- 4.5 microM) and V(max) [1.5 +/- 0.4 units of fluorescence/(100 microg of microsomal protein/mL/s)] were estimated, confirming the metabolism of MI-D and indicating that the reaction follows Michaelis-Menten kinetics. Acute toxicity was established on the basis of an estimation of mean lethal dose (LD-50; 181.2 mg/kg) and histopathological analysis of animals that survived the LD-50 test. Abdominal adhesions, inflammatory foci, and formation of granulomas were observed. Altogether, the results contribute to the advancement of research in support of MI-D as a future chemotherapeutic drug.

Competitive kinetics in free radical reactions of cinnamic acid derivatives. Influence of phenoxyl radicals reactions.

Relative rates of consumption of caffeic, ferulic and sinapic acids by 2,2'-azobis(2-amidine propane) derived peroxyl radicals has been measured in parallel experiments employing a single substrate and in competitive experiments. Rates of consumption measured in independent experiments at low substrate concentrations (first order limit) follow the order: sinapic > ferulic > caffeic. In agreement with this, in competitive experiments employing simultaneously sinapic and caffeic acid the former compound is consumed considerably faster. On the other hand, in competitive experiments employing ferulic and caffeic acids over a wide range of experimental conditions, caffeic acid is consumed considerably faster than ferulic acid, a result that contrasts with that obtained when both compounds are reacted independently. These apparently anomalous results are interpreted in terms of secondary reactions of the phenol-derived radicals. In particular, hydrogen transfer among phenoxyl radicals and the phenols and fast reactions (disproportionation) of caffeic acid derived radicals could explain these discrepancies.

A new sunscreen of the cinnamate class: synthesis and enzymatic hydrolysis evaluation of glyceryl esters of p-methoxycinnamic acid.

Glyceryl esters of p-methoxycinnamic acid, 1,3-dipalmitoyl-2-p-methoxycinnamoyl-1,2,3-propanetriol and 1,3-dioctanoyl-2-p-methoxycinnamoyl-1,2,3-propanetriol were synthesised in an attempt to increase substantivity and decrease eventual undesirable effects of sunscreens of this class. To assess if the glyceryl esters could present a higher stability towards hydrolysis by lipases in the stratum corneum, hydrolysis rates were determined in vitro using a commercial fungal lipase from Rhizomucor miehei. Results presented herein show that the glyceryl esters have similar lambda(max) and epsilon values to sunscreens of the cinnamate class. The ester 1,3-dipalmitoyl-2-p-methoxycinnamoyl-1,2,3-propanetriol presented a 2.8 times lower hydrolysis rate by lipase, in vitro, than the commercial sunscreen 2-ethylhexyl-p-methoxycinnamate (alkyl ester). This finding suggests that this triacylglycerol can possibly have a longer retention time in the skin and consequently promote a more intense and effective antisolar action than the commercial sunscreen.

Development of a transformation system for Crinipellis perniciosa, the causal agent of witches' broom in cocoa plants.

Protoplasts of the pathogenic plant fungus, Crinipellis perniciosa, were transformed to hygromycin B resistance using the pAN7-1 plasmid, which contains the Escherichia coli hph gene under the control of Aspergillus nidulans regulatory sequences. The pAN7-1 plasmid was introduced by PEG/CaCl(2) treatment. Transformation frequencies of 1.6-2.5 transformants/microg of DNA were achieved. About 54% of the transformants were abortive and 40 analyzed transformants were mitotically stable and showed different hygromycin B resistance levels. The presence of the hph gene was checked by PCR in five transformants and the integration of multiple plasmid copies into different genome sites was observed by Southern analysis. This is the first report of a C. perniciosa transformation system and represents an important step for further research into genetic manipulation of this fungal plant pathogen.

Formation of excited states during the oxidation of caffeic and 3,4 dihydroxyphenylacetic acids catalyzed by catechol oxidase.

The oxidation of caffeic and 3,4-dihydroxyphenylacetic acids by catechol oxidase leads to formation of excited species as indicated by chlorophyll sensitized emission. The chemiexcitation step and the transfer are efficient and favoured by the hydrophobic medium. The oxidation of Catecholamines such as L-DOPA, adrenaline and noradrenaline by catechol oxidase does not lead to excitation of chlorophyll.