A highly specific pathogen-responsive promoter element from the immediate-early activated CMPG1 gene in Petroselinum crispum.

Within the complex signalling network from pathogen-derived elicitor perception to defense-related gene activation, some immediate-early responding genes may have pivotal roles in downstream transcriptional regulation. We have identified the parsley (Petroselinum crispum) ELI17 gene as a particularly fast-responding gene possessing a new type of W box-containing, elicitor-responsive promoter element, E17. Highly selective E17-mediated reporter gene expression at pathogen infection sites in transgenic Arabidopsis thaliana plants demonstrated the potential of this promoter element for designing new strategies in resistance breeding as well as for further analysis of the early components of defense-related gene activation mechanisms. The protein encoded by the ELI17 gene exhibits various structural characteristics of established transcription factors and is designated as a CMPG protein according to the first four strictly conserved amino acids defining a newly emerging class of plant-specific proteins.

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley.

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.

[Combined suicide by drinking acetic acid with subsequent hanging]. / Kombinierte Suizide durch Einnahme von Essigsäure mit nachfolgendem Erhängen.

Three late emigrants of German origin from Russia (1 man, 2 women) drank 70- and 86% acetic acid respectively before hanging themselves. In one of the suicides the hanging noose was running across the face. None of the cases presented showed any perioral chemical burns. At autopsy an intense odor of acetic acid was noticeable. The mucosa of the upper digestive tract showed a whitish, grey, brown or black discoloration and was partially detached. In two cases intravascular clotting of blood was discernible in the area affected by the acid; in one case the vessels were imbibed with haemoglobin due to haemolysis. Highly concentrated acetic acid is generally available in the successor states of the former USSR and its use in suicides is thus not uncommon. The legal regulations applying in Germany to the sale of acetic acid and the toxicological data are briefly described.

Regulation and functional expression of cinnamate 4-hydroxylase from parsley.

A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10). Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley. The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues. These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit.

Extensive reprogramming of primary and secondary metabolism by fungal elicitor or infection in parsley cells.

The transcription rates of numerous plant genes have previously been shown to be strongly affected by pathogen infection or elicitor treatment. Here we estimate the extent and complexity of this response by analyzing the patterns of mRNA induction in fungal elicitor-treated parsley cells (Petroselinum crispum) for several representatives from various primary and secondary metabolic pathways, cytosolic as well as plastidic. As a reference, we use the biphasic accumulation curve for the coordinately induced mRNAs encoding the three core enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and 4-coumarate:CoA ligase. Coincidence with this curve was observed for the mRNA induction kinetics of several, but not all, phenylpropanoid branch pathway-related reactions, whereas seven selected mRNAs from the pentose phosphate, glycolytic and shikimate pathways, including various cytosolic and plastidic isoforms, were induced with great differences in timing. Likewise unique and dissimilar from the reference curve were the induction patterns for various mRNAs encoding enzymes or proteins that are either more distantly or not at all related to phenylpropanoid metabolism. None of over 40 mRNAs tested so far remained unaffected. Using one strongly elicitor-responsive mRNA from carbohydrate metabolism, encoding a cytosolic glucose 6-phosphate dehydrogenase, for in situ RNA/RNA hybridization in fungus-infected parsley leaf tissue, we observed again the previously reported, close simulation of metabolic changes in true plant/fungus interactions by elicitor treatment of cultured cells. In addition to demonstrating extensive, highly complex functional, temporal and spatial patterns of changes in gene expression in infected plant cells, these results provide valuable information for the identification of pathogen-responsive promoters suitable for gene technology-assisted resistance breeding.

[A case of suicidal chloroform poisoning]. / Zur Kasuistik der suizidalen Chloroformintoxikation.

A 33-year-old man who had suffered from phobia and depressions was found dead in his kitchen. He was entirely covered by plastic waste disposal bags stuck together. The corpse was lying flat on his stomach with his face on a towel soaked with chloroform. Within the plastic cover, on either side of the body's head, there was located a can half way filled with chloroform. Autopsy revealed cauterized lips and mucous membranes of the mouth. The morphological findings of the inner organs were unspecific. For toxicological analyses, air samples from the pleural cavities of the corpse were taken by way of charcoal tubes and a microprocessor aided pump. The solvents adsorbed on the charcoal were desorbed with benzyl alcohol and analyzed by gas chromatography according to standard procedures. The quantification of the chloroform levels of the body fluids and the tissue samples of the corpse was performed by extraction with pentane followed by addition of trichloroethylene as internal standard and consecutive gas chromatographic analysis. The results of the toxicological analyses confirmed the diagnosis of a fatal chloroform intoxication.

A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

[Suicide or fatal accident in fantasy play?]. / Suizid oder tödlicher Unfall bei Fantasy-Spiel?

The death by drowning of a 25-year-old man is reported, whose putrescent body was found drifting on the surface of an artificial lake. The deceased was dressed in a coat of chain-mail and trousers of the same material. The chain-mail clothing, which had a weight of 20 kg, reminded one of a knight's armour; it had been made by the wearer himself. Around the neck there were several turns of a chain running down to the legs and tying them together. According to the police investigations it is to be assumed that the man went out on the lake in a rubber boat and then pierced the air chambers of the boat with a knife. The possible background of the case in context with the special liking for fantasy games is discussed.

[Accidental CO poisoning by operating a charcoal grill]. / Akzidentelle CO-Vergiftung durch Betrieb eines Holzkohlegrills.

In the hut of a garden plot a male body of at first unknown identity was found. Although a natural death was assumed, autopsy was ordered by the court because of the unclear identity. On autopsy bright red post-mortem lividity was found; an assay of the blood showed 55% CO-Hb. A second inspection of the place, where the body was found, revealed that a garden grill, which had originally stood near the entrance of the hut, had been carried out into the garden. To reconstruct the case, measurements of the carbon monoxide concentration in the air of the room were carried through after starting the charcoal grill. The results showed that within 60 min. the carbon monoxide content rose to 0.21% by volume and thus clearly exceeded the limit of 0.1% by volume considered as lethal.

Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes.

The effects of UV light or fungal elicitors on plant cells have so far been studied mostly with respect to defense-related gene activation. Here, an inverse correlation of these stimulatory effects with the activities of several cell cycle-related genes is demonstrated. Concomitant with the induction of flavonoid biosynthetic enzymes in UV-irradiated cell suspension cultures of parsley (Petroselinum crispum), total histone synthesis declined to about half the initial rate. A subclass of the histone H3 gene family was selected to demonstrate the close correlation of its expression with cell division, both in intact plants and cultured cells. Using RNA-blot and run-on transcription assays, it was shown that one arbitrarily selected subclass of each of the histone H2A, H2B, H3 and H4 gene families and of the genes encoding a p34cdc2 protein kinase and a mitotic cyclin were transcriptionally repressed in UV-irradiated as well as fungal elicitor-treated parsley cells. The timing and extent of repression differed between the two stimuli; the response to light was more transient and smaller in magnitude. These differential responses to light and elicitor were inversely correlated with the induction of phenylalanine ammonia-lyase, a key enzyme of phenylpropanoid metabolism. Essentially the same result was obtained with a defined oligopeptide elicitor, indicating that the same signaling pathway is responsible for defense-related gene activation and cell cycle-related gene repression. A temporary (UV light) or long-lasting (fungal elicitor) cessation of cell culture growth is most likely due to an arrest of cell division which may be a prerequisite for full commitment of the cells to transcriptional activation of full commitment of the cells to transcriptional activation of pathways involved in UV protection or pathogen defense. This conclusion is corroborated by the observation that the histone H3 mRNA level greatly declined around fungal infection sites in young parsley leaves.

[Detection of metal traces in electric marks]. / Zum Nachweis von Metallspuren in Strommarken.

Report on a suicidal electrocution, in which a 29-year-old man twisted copper wires around his wrists fixing the ends to an emergency current aggregate. Heavy metal analyses of the electrical marks were performed with ICP-AES-spectrometry.

Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley.

We describe a complete gene family encoding phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in one particular plant species. In parsley (Petroselinum crispum), the PAL gene family comprises two closely related members, PAL1 and PAL2, whose TATA-proximal promoter and coding regions are almost identical, and two additional members, PAL3 and PAL4, with less similarity to one another and to the PAL1 and PAL2 genes. Using gene-specific probes derived from the 5' untranslated regions of PAL1/2, PAL3, and PAL4, we determined the respective mRNA levels in parsley leaves and cell cultures treated with UV light or fungal elicitor and in wounded leaves and roots. For comparison, the functionally closely related cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL) mRNAs were measured in parallel. The results indicate various degrees of differential responsiveness of PAL4 relative to the other PAL gene family members, in contrast to a high degree of coordination in the overall expression of the PAL, C4H, and 4CL genes. The only significant sequence similarities shared by all four PAL gene promoters are a TATA-proximal set of three putative cis-acting elements (boxes P, A, and L). None of these elements alone, or the promoter region containing all of them together, conferred elicitor or light responsiveness on a reporter gene in transient expression assays. The elements appear to be necessary but not sufficient for elicitor- or light-mediated PAL gene activation, similar to the situation previously reported for 4CL.

Oligopeptide elicitor-mediated defense gene activation in cultured parsley cells.

We have used suspension-cultured parsley cells (Petroselinum crispum) and an oligopeptide elicitor derived from a surface glycoprotein of the phytopathogenic fungus Phytophthora megasperma f.sp. glycinea to study the signaling pathway from elicitor recognition to defense gene activation. Immediately after specific binding of the elicitor by a receptor in the plasma membrane, large and transient increases in several inorganic ion fluxes (Ca2+, H+, K+, Cl-) and H2O2 formation are the first detectable plant cell responses. These are rapidly followed by transient changes in the phosphorylation status of various proteins and by the activation of numerous defense-related genes, concomitant with the inactivation of several other, non-defense-related genes. A great diversity of cis-acting elements and trans-acting factors appears to be involved in elicitor-mediated gene regulation, similar to the apparently complex nature of the signal transduced intracellularly. With few exceptions, all individual defense responses analyzed in fungus-infected parsley leaves have been found to be closely mimicked in elicitor-treated, cultured parsley cells, thus validating the use of the elicitor/cell culture system as a valuable model system for these types of study.

Structural and catalytic properties of the four phenylalanine ammonia-lyase isoenzymes from parsley (Petroselinum crispum Nym.).

Near-full-length cDNAs for the four phenylalanine ammonia-lyase (PAL) isoenzymes in parsley (Petroselium crispum Nym.) were cloned and the complete amino acid sequences deduced. Fusion proteins with glutathione S-transferase were expressed in Escherichia coli, purified and cleaved. All of the resulting phenylalanine ammonia-lyase proteins, as well as the fusion proteins, were catalytically active. The turnover number of one selected isoenzyme, PAL-1, was estimated to be around 22 s-1 for each active site. In contrast to a certain degree of differential expression in various parts of parsley plants, the four phenylalanine ammonia-lyase isoenzymes exhibited very similar apparent Km values for L-phenylalanine (15-24.5 microM) as well as identical temperature (58 degrees C) and pH (8.5) optima. All of them were competitively inhibited by (E)-cinnamate with similar efficiency (Ki values: 9.1-21.5 microM), lacked cooperative behaviour, and accepted L-tyrosine as a substrate with low affinity (Km values: 2.6-7.8 mM). These results suggest that the occurrence of multiple gene copies has a function other than encoding isoenzymes with different enzyme kinetic properties.

alpha-Tocopherol supplementation in racing cyclists during extreme endurance training.

This study was undertaken to evaluate the effects of 5 months of alpha-tocopherol supplementation on physical performance during aerobic exercise training in 30 top-class cyclists. Antioxidative effects of supplementation were also studied. Plasma alpha-tocopherol concentration increased significantly in the vitamin E-supplemented group, whereas the placebo group showed a trend toward decrease. Physical performance did not improve in the alpha-tocopherol-supplemented group compared to the placebo group. Heart rates were also not significantly different. Lactate concentrations at the aerobic threshold and the anaerobic threshold were identical. Thus, there was no performance improvement in the alpha-tocopherol-supplemented group. However there was a significant reduction in CK in serum of the E-supplemented group. A trend toward decrease in GOT, GPT, and LDH was observed with alpha-tocopherol supplementation. Moreover, significantly reduced malondialdehyde serum levels were measured in the E-supplemented group. The findings indicate a protective effect of alpha-tocopherol supplementation against oxidative stress induced by strenuous exercise.

Lipid peroxidation and antioxidative vitamins under extreme endurance stress.

The aim of this study was to examine whether extreme endurance stress of trained athletes can influence lipid peroxidation and muscle enzymes. A randomized and placebo-controlled study was carried out on 24 trained long-distance runners who were substituted with alpha-tocopherol (400 I.U. d-1) and ascorbic acid (200 mg d-1) during 4.5 weeks prior to a marathon race. The serum concentrations of retinol, ascorbic acid, beta-carotene, alpha-tocopherol, malondialdehyde (TBARS) and uric acid as well as glutathione peroxidase (GSH Px) and catalase were measured 4.5 weeks before (A), immediately before (B), immediately after (C) and 24 h after (D) the course. After competition (C) TBARS serum concentrations of the athletes (n = 22) decreased in both groups (P < 0.0001). The ascorbic acid serum concentration increased significantly in the supplemented group from (A) to (B) (P < 0.01), from (B) to (C) (P < 0.001) and in the placebo group a significant increase from (B) to (C) (P < 0.01) was observed. The alpha-tocopherol serum concentration increased significantly in the supplemented group from (A) to (B) (P < 0.001) and from (B) to (C) (P < 0.05). The enzymes glutathione peroxidase (GSH Px) and catalase measured in erythrocytes as well as the serum selenium levels did not show significant differences at any time. A significant increase of CK concentration was observed from (C) to (D) in the supplemented group (P < 0.01) and in the placebo group (P < 0.001). The increase of CK serum concentration is remarkably lower in the supplemented group compared with the placebo group (P < 0.01). It is concluded that endurance training coupled with antioxidant vitamin supplementation reduces blood CK increase under exercise stress.

[Selenium metabolism and glutathione peroxidase activity of endurance athletes in rest and under exertion]. / Die Selenversorgung und die Glutathionperoxidaseaktivität von Ausdauerathleten in Ruhe und unter Belastung.

Selenium which has been recognised as an essential trace element for mammals and humans is part of the enzyme glutathione peroxidase (GSHPx) and of other selenium-dependent enzymes. GSHPx protects the organism from oxidative damage. Supposing that the formation of peroxides is enhanced in athletes during a marathon run the question arose whether the selenium concentrations in the blood plasma and the GSHPx activities in the erythrocytes are changed. Our experiments were performed with 13 athletes completing a marathon. Blood probes were taken before, immediately after and two hours after the endurance event. No significant changes in the selenium concentrations of the blood plasma probes nor of the enzyme glutathione peroxidase activities of the erythrocytes were observed during the experiments. The selenium concentrations in the plasma: 77.7 +/- 20.8 micrograms/l and the GSHPx activities of the erythrocytes 18.2 +/- 7.4 U/g Hb were within normal clinical range. Symptoms of selenium deficiency could not be detected. In order to answer the question of whether the selenium concentrations in the blood of the athletes represent an optimal level for preventing enhanced formation of peroxides, intervention studies are needed, including measurement of more parameters of lipid peroxidation.

[Postmortem diagnosis of exogenous insulin administration]. / Zur postmortalen Diagnostik der exogenen Insulin-Applikation.

Toxicological analyses are presented in connection with the decrease of a 51-year-old female medical practitioner. The woman was found dead in a woodland about 250 metres far from her motor car. According to her relatives the woman had expressed her intention to commit suicide. Two single use syringes, two empty (300 units) cartridges intermediate type human insulin, and one empty ampoule of 10 mg diazepam were found near the corpse. The toxicological analyses of the heart-blood resulted in 210 ng diazepam/ml, 382 mu units/ml insulin and 0.58 ng/ml C-peptide. After extraction of a tissue sample from the injection area (cubital region) 113 milliunits insulin could be detected by radioimmunoassay. The insulin concentration in relation to the C-peptide concentration in the blood of the corpse indicated an exogenous supply of insulin. The HbA1c and fructosamine data in the blood and the histological examination of the pancreas showed that the woman had not suffered from diabetes.

[Poisoning caused by a paint stripping agents containing dichloromethane]. / Intoxikation mit einem Dichlormethan-haltigen Abbeizmittel.

It is widely known that inhalation of the solvent dichloromethane can lead to severe intoxication. We report on a fatal accident of a 29-year-old painter. He had to remove the old chlorocaoutchoucpaint of two overflow pools adjoining an empty swimming pool by sand-blasting. After obviously having finished his work in the first pool the painter was found dead, lying on the floor of the second pool. Later an almost empty 10-kg-canister of a paint stripper (macerating agent) the main component of which was dichloromethane was found in the car of the painter. Blood samples of the corpse measured by head space gas chromatography and electron capture detection showed dichloromethane levels in the range of 513-773 micrograms/ml.