Influence of the developmental stage on the (proto)-hypericin and (proto)pseudohypericin levels of Hypericum plants from Crete.

The contents of (pseudo)hypericin and their immediate precursors were studied in wild populations of various Hypericum species on the island of Crete, Greece. Therefore, the aerial parts of wild grown H. perforatum, H. triquentrifolium, H. empetrifolium and H. perfoliatum shoots were collected throughout the island and the quantitative variations in (proto)hypericin and (proto)pseudohypericin examined. The plant material was harvested at different stages of the life cycle of the species and the contents in the above-mentioned compounds analyzed discriminating between flowers/fruits and leaves/petioles. HPLC analysis of hypericin, pseudohypericin and their immediate precursors, protohypericin and protopseudohypericin, revealed great differences in the contents of the compounds in dependence on the developmental stage of the plants. In all examined species the highest concentrations of hypericin were found during blossoming whereas the lowest concentrations were present during ripening of the fruits. H. perforatum and H. triquentrifolium show much higher hypericin levels in flowers/fruits compared to leaves/petioles, whereas the species H. empetrifolium and H. perfoliatum show similar concentrations of total hypericins in both flowers/fruits and leaves/petioles. In the different species the levels of (proto)hypericin and (proto)pseudohypericin varied, but in almost all samples from flowers/fruits and leaves/petioles the ratio of (proto)hypericin to (proto)pseudohypericin was higher than one. When the total amount of hypericins per entire aerial part of a plant was calculated for all developmental stages, we found that H. perforatum contained the highest amount of hypericin. This in combination with the comparatively high concentration of hypericins in flowers/fruits and in leaves/petioles in this species, as well as the high ratio of (proto)hypericin to (proto)pseudohypericin, especially during the developmental stage of blossoming, encourages us to think about the possibility of cultivating Hypericum perforatum in Crete as a medicinal plant in the future.

Changes in the LHCII-mediated energy utilization and dissipation adjust the methanol-induced biomass increase.

Considerably low methanol concentrations of 0.5% (v/v), induce an immense increase in biomass production in cultures of the unicellular green alga Scenedesmus obliquus compared to controls without additional methanol. The effect is light-regulated and it mimics high-CO2 induced changes of the molecular structure and function of the photosynthetic apparatus. There is evidence that methanol enhances under high light conditions by molecular changes in the LHCII--a decrease of the functional antenna-size per active reaction center--the photochemical effectiveness of the absorbed energy. This means that the non-photochemical quenching (NPQ) is minimized and thereby the overall dissipation energy. Experiments with mutants of Scenedesmus Wt produced evidence that the LHCII is the locus of the mechanism which regulates the methanol effect. The employed mutants were Wt-LHC, lacking a functioning LHCII, the light-dependent greening mutant C-2A', and the double mutant C-2A'-LHC, combining both mutations.

Light-dependent induction of strongly increased microalgal growth by methanol.

Low methanol concentrations (about 0.5% v/v) induce biomass production in cultures of the unicellular green alga Scenedesmus obliquus by more than 300%, compared to controls without this solvent. This effect on the microalgal growth was found to be dependent on the solvent concentration, the packed cell volume (PCV), light intensity and light quality. It could be shown that methanol addition leads to a decrease in size of the light harvesting complex (LHC) on the basis of chlorophylls and proteins, and thus to changes in structure and functioning of the photosynthetic apparatus. These alterations lead to enhanced photosynthesis and respiration rates. The action of methanol on the photosynthetic apparatus is thus comparable to the effect of enhanced CO(2) concentrations. These findings support the previously proposed pathway for methanol metabolization with CO(2) as the final product. We conclude that the subsequent assimilation of the increased CO(2) amounts by the Calvin-Benson cycle is a possible explanation for the methanol-mediated increase in biomass production in terms of PCV. The methanol effect is observed only in the light and in the presence of a functioning photosynthetic apparatus. Preliminary action spectra suggest that the primary photoreceptor is a chlorophyll-protein complex with two absorption maxima at 680 and 430 nm, which may possibly be attributed to the reaction center of photosystem II (PSII).

DNA damage by 3,6-dihydropyrazine-2,5-dipropanoic acid, the cyclic dimerization product of 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in lead poisoning and inborn porphyrias. It has been shown to produce reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes, DNA, and subcellular structures. Studies have also shown that ALA may condense to yield the cyclic product 3,6-dihydropyrazine-2,5-dipropanoic acid (DHPY). Here we propose that DHPY could be involved in DNA damage in the presence of high concentrations of ALA. Exposure of plasmid pUC19 DNA to low concentrations of DHPY (2-10 microM) in the presence of 0.1 mM Cu2+ ions causes DNA strand breaks, as demonstrated by agarose gel electrophoresis. It was also shown that in the presence of Cu2+ ions DHPY is able to increase the oxidation of monomeric 2'-deoxyguanosine to form 8-oxo-7,8-dihydro-2'-deoxyguanosine as inferred from high performance liquid chromatography measurements using electrochemical detection. Addition of a metal chelator (bathocuproine, 0.5 mM), the DNA compacting polyamines spermidine (1 mM) and spermine (1 mM) or antioxidant enzymes such as superoxide dismutase (10 microg/ml) and catalase (20 pg/ml) protect the DNA against these damages. The data presented here are discussed with respect to the increased frequency of liver cancer in patients with acute intermittent porphyria.

DNA damage by 5-aminolevulinic and 4,5-dioxovaleric acids in the presence of ferritin.

Cellular accumulation of 5-aminolevulinic acid (ALA), the first specific intermediate of heme biosynthesis, is correlated in liver biopsy samples of acute intermittent porphyria affected patients with an increase in the occurrence of hepatic cancers and the formation of ferritin deposits in hepatocytes. 5-Aminolevulinic acid is able to undergo enolization and to be subsequently oxidized in a reaction catalyzed by iron complexes yielding 4,5-dioxovaleric acid (DOVA). The released superoxide radical (O(*-)(2)) is involved in the formation of reactive hydroxyl radical ((*)OH) or related species arising from a Fenton-type reaction mediated by Fe(II) and Cu(I). This leads to DNA oxidation. The metal catalyzed oxidation of ALA may be exalted by the O(*-)(2) and enoyl radical-mediated release of Fe(II) ions from ferritin. We report here the potentiating effect of ferritin on the ALA-mediated cleavage of plasmid DNA and the enhancement of the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo). Plasmid pBR322 was incubated with ALA and varying amounts of purified ferritin. DNA damage was assessed by gel electrophoresis analysis of the open and the linear forms of the plasmid from the native supercoiled structure. Addition of either the DNA compacting polyamine spermidine or the metal chelator ethylenediaminetetraacetic acid (EDTA) inhibited the damage. It was also shown that ALA in the presence of ferritin is able to increase the oxidation of the guanine moiety of monomeric 2'-deoxyguanosine (dGuo) and calf thymus DNA (CTDNA) to form 8-oxodGuo as inferred from high performance liquid chromatography (HPLC) measurements using electrochemical detection. The formation of the adduct dGuo-DOVA was detected in CTDNA upon incubation with ALA and ferritin. In a subsequent investigation, the aldehyde DOVA was also able to induces strand breaks in pBR322 DNA.

Measurement of 4,5-dioxovaleric acid by high-performance liquid chromatography and fluorescence detection.

In this work we describe a sensitive method for the detection of 4,5-dioxovaleric acid (DOVA). 4,5-Dioxovaleric acid is derivatized with 2,3-diaminonaphthalene to form 3-(benzoquinoxalinyl-2)propionic acid (BZQ), a product with favorable UV absorbance and fluorescence properties. The high-performance liquid chromatographic method with UV absorbance and fluorescence detection is simple and its detection limit is approximately 100 fmol. This method was used to detect 4,5-dioxovaleric acid formation during metal-catalyzed 5-aminolevulinic acid (ALA) oxidation. Iron and ferritin were active in the formation of 4,5-dioxovaleric acid in the presence of 5-aminolevulinic acid. In addition, HPLC-MS-MS assay was used to characterize BZQ. The determination of 4,5-dioxovaleric acid is of great interest for the study of the mechanism of the metal-catalyzed damage of biomolecules by 5-aminolevulinic acid. This reaction may play a role in carcinogenesis after lead intoxication. The high frequency of liver cancer in acute intermittent porphyria patients may also be due to this reaction.

Purification, metal cofactor, N-terminal sequence and subunit composition of a 5-aminolevulinic acid dehydratase from the unicellular green alga Scenedesmus obliquus, mutant C-2A'.

5-Aminolevulinic acid dehydratase was purified to apparent homogeneity from Scenedesmus obliquus, mutant C-2A', starting with serial affinity chromatography according to Wang et al., followed by separation on DEAE-Cellulose DE 52, TSKgel Toyopearl HW-55 and FPLC on Mono Q. The enzyme was purified 117-fold compared with the initial crude soluble enzyme preparation and showed a final specific activity of 9.17 microkat/kg protein at pH 8.2 at a total recovery of 7%. Mg2+ was determined to be the metal cofactor of the enzyme. It can, to a certain extent, be substituted by other divalent cations. From the purified enzyme the first 15 amino acids of the N-terminus could be determined, showing a moderate similarity to 5-aminolevulinic acid dehydratases from spinach, pea, Escherichia coli and yeast. The molecular mass of the native protein was determined by gel filtration to be 282+/-5 kDa. 42+/-1 kDa were ascertained for the subunit size by SDS/PAGE. These investigations, supported by electron microscopy, revealed that the enzyme from Scenedesmus consists of six subunits arranged in a six-membered ring. Additionally, there is some evidence that two of the rings form a sandwich-like complex.

Purification, characterization and N-terminal sequence of phosphoserine aminotransferase from the green alga Scenedesmus obliquus, mutant C-2 A'.

Phosphoserine aminotransferase (EC 2.6.1.52), an enzyme of the "phosphorylated pathway" leading to the formation of serine, was purified from Scenedesmus obliquus, mutant C-2 A'. Purification started from the soluble supernatant of a crude cell homogenate and included different affinity and DEAE chromatographic techniques, as well as gel filtration. The purified phosphoserine aminotransferase was enriched 1537-fold and identified to be a homodimer with subunit molecular masses of 40 kDa, each. The absorption spectrum is consistent with the presence of pyridoxal-5-phosphate as cofactor. From the purified enzyme 18 amino acids of the N-terminus could be determined, showing at least 67% homology with the serC gene encoding phosphoserine aminotransferases from bacterial organisms.

Formation of 5-aminolevulinate via glutamate-1-semialdehyde and 4,5-dioxovalerate with participation of an RNA component in Scenedesmus obliquus mutant C-2A'.

In the yellow mutant C-2A' of the unicellular green alga Scenedesmus obliquus the participation of an RNA species in the conversion of glutamate to 5-aminolevulinate is clearly demonstrated by the fact that RNAase treatment of a soluble enzyme preparation drastically decreases the formation of 5-aminolevulinate. The involvement of 4,5-dioxovalerate in the C5 pathway is demonstrated by the decrease of label in enzymatically formed 5-aminolevulinate from [14C]glutamate by providing an increased unlabelled pool of 4,5-dioxovalerate. Evidence supporting the role of glutamate-1-semialdehyde as an additional intermediate in the reaction sequence is also presented. We propose a new reaction scheme, consistent with the results reported here, for the formation of 5-aminolevulinate via the C5 pathway.

The synthesis and properties of 4,5-dioxovaleric acid, a possible intermediate in the biosynthesis of 5-aminolaevulinic acid, and its in vivo formation in Scenedesmus obliquus.

The formation of 5-aminolaevulinic acid for chlorophyll biosynthesis in the pigment mutant C-2A' of the unicellular green alga Scenedesmus obliquus occurs via two pathways: the first and major pathway uses glycine and succinyl-CoA as precursors and the second, the C-5 pathway, uses the intact five C-atom skeleton of either glutamate or 2-oxoglutarate. The intermediates in this latter pathway of 5-aminolaevulinic acid biosynthesis are still a matter of controversy and discussion but, in this paper, we have demonstrated in this Scenedesmus mutant that 4,5-dioxovaleric acid occurs in the cells when illuminated in the presence of laevulinic acid and the amount of 4,5-dioxovaleric acid formed is dependent on the period of illumination. In a preliminary experiment we have shown, under similar conditions, that labelled 4,5-dioxovaleric acid can be obtained from both DL-[1-(14)C]glutamate and [2-(14)C]glycine. This suggests that 4,5-dioxovaleric acid is formed enzymically from the intact five C-atom skeleton of glutamate but may also arise from the deamination of ALA formed by the condensation of succinyl-CoA and glycine by the 5-aminolaevulinic acid synthase pathway. To obtain positive identification the naturally occurring, and also the labelled, 4,5-dioxovaleric acid were isolated as the more stable benzoquinoxaline derivative by condensation with 2,3-diaminonaphthalene and identified by comparison with the benzoquinoxaline derivative of an authentic sample of 4,5-dioxovaleric acid prepared by the hydrogenation of the ozonide of benzylidene laevulinic acid. The structures of the authentic sample of 4,5-dioxovaleric acid and its benzoquinoxaline derivative were confirmed by NMR and mass spectroscopy and both were further characterized by TLC and by infrared, ultraviolet and fluorescence spectroscopy.