Volume 41, Issue 9-10

It is shown that 2-hydroxy-4-methoxy-5-methyl pyridine N-oxide isolated from Pseudomonas cepacia which is known as a siderophore has a comparable complexing capacity for Al 3+ .

Heterotrophic cell suspension cultures of Ruta graveolens were established by reversion of photomixotrophic cultures without any change in the chemical composition of the growth medium. The heterotrophic cultures were qualitatively and quantitatively analyzed by gas- chromatography and mass spectroscopy for volatile compounds. The terpenoid hydrocarbons geijerene and pregeijerene, C 6 -C 8 ketones, acetic acid n-butylester and a series of aliphatic C 4 -C 9 primary and secondary alcohols were found as main constituents. Two isomeric sabinene hydrates were also isolated as new constituents of rue cells. The data are discussed in comparison to results obtained with photomixotrophic cell suspension cultures.

1.The flavonoid constituents of Flaveria bidentis have been identified as the 3-O-glucosides of kaempferol. 6-methoxykaempferol and 6-methoxyquercetin (patuletin), as well as six flavonol sulfate esters belonging to quercetin and isorhamnetin. The two latter glucosides are reported here for the first time in this species; whereas quercetin-3,7-disulfate, is a novel compound from nature. 2.The amounts of both glucosides and sulfate esters in young seedlings were highest in buds > stems > leaves on fresh weight basis, whereas they were undetectable in the root. There were striking quantitative differences in the flavonoid pattern of young seedlings, as compared with mature shoots, especially the flavonol sulfate esters which were predominant in seedlings. 3.The biosynthesis of both groups of flavonoids from [ 3 H]cinnamate and [ 35 S]sulfate showed that the former was predominantly incorporated into glucosides whereas the latter, into sulfate esters; thus indicating that sulfation is a later step in the biosynthesis of flavonol sulfate esters. 4.The significance of the glucosylation and sulfation reactions was discussed in relation to the regulation of biosynthesis and compartmentation of both groups of compounds.

Plectonem a, Arylsulfatase. Thiosulfate R eductase, Substrate Induction, Cyanobacteria Arylsulfatase activity of the cyanobacterium P lectonem a 73110 is detected during growth on p-nitrophenyl sulfate as only sulfur source. A 60-fold purification of the soluble arylsulfatase yields a protein with an pH-optim um around 10 and K m data for p-nitrophenyl sulfate of 9.5 × 10" 5 and for nitrocatechol sulfate of 3.4 × 10-5. The enzym atic activity is not effected by the cations Mg 2- , M n 2+ , K 2+ or Na + used as chloride salts. The anions SO 3 2- and SO 4 2- did not inhibit this activity, how ever EDTA , thiols and triton X -100 decreased this activity to about 25% w here­ as SDS inhibited com pletely. Sulfate limitation is necessary for the developm ent of arylsulfatase activity in the presence of its substrate p-nitrophenyl sulfate. This arylsulfatase activity is partly bound to m embranes.

The accumulation of volatile metabolites in liquid cultures of the basidiomycete Gloeophyllum odoratum CBS 444.61 was studied after cultivation on defined synthetic liquid culture medium containing glucose (2%), asparagine (0.15%), and mineral salts. The composition of the steam volatile compounds differed distinctly from that obtained with another, previously investigated strain of this species. Two major constituents, the bicyclic sesquiterpene alcohol drimenol and a linalool derivative, 3,7-dimethyl-3-hydroxy-6-octenic acid methyl ester, could be identified by mass spectral data and 1 H and 13 C NMR spectroscopy. Besides several monoterpene alcohols, other minor constituents were trans-linalool oxide and 1-octen-3-ol. 3,7-Dimethyl-3-hydroxy-6-octenic acid methyl ester is described for the first time as a natural product.

Several biotic and abiotic clicitors of the hypersensitive reaction in wheat leaves are described. The elicitors induce enhanced activity of phenylalanine ammonia-lyase with subsequent lignifica­tion, visible as a yellow autofluorescence. The deposited material stains positively with phloroglucinol. DEAE-dextran, epoxystearic acid and chitosan are strong elicitors, while the glucans tested have no activity. A biotic elicitor (genuine Pgt-elicitor) was isolated from the germ tube walls of uredospores of Puccinia graminis f. sp. tritici. The high molecular weight, water soluble elicitor is heat stable and unaffected by mild acid and mild alkaline treatments. It seems to be a glycoprotein with the carbohydrate moiety bearing the active residues, as indicated by periodate sensitivity and pro­tease stability. The carbohydrate moiety consists mainly of glucose with some galactose and mannose. Upon ultrafiltration and gel chromatography elicitor activity was associated with fractions of molecular weight of more than 100 kD. When injected into the intercellular space of primary wheat leaves, the elicitor induces lignifica­tion, preceded by an increase in phenylalanine ammonia-lyase activity.

The genuine biotic elicitor from germ tube walls of Puccinia graminis f. sp. tritici (Pgt-elicitor) induces lignification, preceded by an increase in phenylalanine ammonia-lyase (PAL) activity, when injected into the intercellular space of primary wheat leaves. This increase in PAL activity is accompanied by an increase in other enzyme activities of the general phenylpropanoid pathway and the specific pathway of lignin biosynthesis: 4-coumarate :CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (PO). Total extractable activities of 4CL, CAD and PO do not differ significantly in resistant and susceptible near isogenic wheat lines, whereas the dose response curve of induced PAL activity shows significantly higher values in the resistant isoline. This difference is not observed after injection of other biotic and abiotic elicitors of the lignification response. Although total induced PO activities 24 h after elicitor treatment are equal in both isolines, the PO isoenzyme pattern of resistant plants differs markedly from that of susceptible plants. The patterns of PO isoenzymes in the compatible and incompatible interaction 48 h after inoculation with Puccinia graminis f. sp. tritici also show differences, which closely resemble those seen after injection of the Pgt-elicitor. The patterns observed after injection of an artificial biotic elicitor clearly differ from those of the natural interaction.

The trichothecene mycotoxin 3-acetyldeoxynivalenol was transformed by cultures of the diace- toxyscirpenol producer Fusarium sp. strain C37410-90 into four compounds, identified as deoxy- nivalenol, 15-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol and fusarenon-X. The major transformations are the result of specific esterification and de-esterification processes, but the production of fusarenon-X involves in addition a novel 4p-hydroxylation of the trichothecene ring system itself.

Ethylmethylglyoxal bis(guanylhydrazone) (EMGBG) sulfate, an analog of the well-known antileukemic drug methylglyoxal bis(guanylhydrazone), was synthesized. It was shown to be an extremely powerful competitive inhibitor of eukaryotic S-adenosyimethionine decarboxylase, with an apparent K i value 12 nᴍ. Thus, it appears to be the most powerful known inhibitor of the enzyme, being almost an order of magnitude more powerful than the corresponding ethylglyoxal derivative. It neither inhibited the proliferation of mouse L1210 leukemia cells in vitro, nor did it potentiate the growth inhibition produced by α-difluoromethyl ornithine. In this respect, its properties are closely related to those of dimethylglyoxal, ethylglyoxal and propylglyoxal bis(guanylhydrazones), while in striking contrast to those of the antiproliferative glyoxal and methylglyoxal analogs. EMGBG also inhibited intestinal diamine oxidase activity (K i 0.7 μᴍ). EMGBG sulfate was crystallized from water, giving orthorhombic crystals (space group Pbcn). Their crystal and molecular structure was determined by X-ray diffraction methods. The carbon-nitrogen double bonds between the ethylmethylglyoxal part and the aminoguanidine moieties were found to have the same configuration as they are known to have in the salts of glyoxal. methylglyoxal and propylglyoxal bis(guanylhydrazones). The glyoxal bis(guanylhydrazone) chain of the EMGBG cation deviated strongly from planarity, thus differing dramatically from the corresponding chains of the glyoxal, methylglyoxal and propylglyoxal analogs.

Soluble protein kinase and protein phosphatase activities were localized in the cytosol of pea mesophyll cells using protoplasts fractionation techniques. The molecular weights of the phosphorylated cytosolic proteins, as determined by polyacrylamide gel electrophoresis, were 68, 55, 46, 38, 36, 30, 22 and 12 kDa. Histone and, to a much lesser extent, casein but not phosvitin were accepted as exogenous substrates. In every case serine served as acceptor amino acid for the phosphate residue. The protein phosphorylation activity had an alkaline pH optimum, and showed no response to varying Mg -2 , Ca 2+ , P i cyclo-AMP or calmodulin concentrations. The kinase activity was competitively inhibited by ADP and pyrophosphate with apparent K i values of 0.5 and 0.17 mᴍ , respectively. High ATP concentrations (1-4 mᴍ) resulted in a strong decrease of radioactivity in the 32 P labeled proteins. It is proposed that the ratio of protein phosphorylation to protein dephosphorylation is regulated by the ATP to ADP ratio in the cytosol.

Diethyldithiocarbamate (DEDTC) does not accept electrons from the photosynthetic electron transport (PET) but can donate electrons to a photosystem I (PSI) Mehler reaction in the pres­ence of the following PET inhibitors: DCMU. DBMIB, and bathophenanthroline. It cannot photoreduce PSI in the presence of cyanide, a PET inhibitor. These data indicate that the site of electron donation is after the plastoquinone pool. Ascorbate is not required for the ability of DEDTC to donate electrons to PSI. There is no photoreductant activity by DEDTC inferredoxin/NADP Hill reactions. Superoxide dismutase inhibits DEDTC/DCMU or bathophenanthroline→methylviologen/O 2 Mehler reaction. Catalase does not recover the consumed O 2 from a DEDTC/DCMU→methylviologen/O 2 Mehler reaction, indicating O 2 - has not been dismutating into H 2 O 2 . These results indicate that superoxide is required for DEDTC ability to donate electrons, therefore DEDTC is limited only to Mehler-type reactions.

During growth of Propionibacterium acnes on Eagles medium protoporphyrin was accumulated inside the cells and coproporphyrin, both as a free base as metalcontaining, outside the cells. The photochemical processes in the endogenous porphyrins were studied by fluorescence spectroscopy during continuous irradiation of Propionibacterium acnes in suspension. The irradiation caused initially an increase in the content of protoporphyrin in the cells in comparison to that which had been accumulated during growth. Maximum light induced protoporphyrin production was achieved in 5 days old cultures. In old cultures where there was practically no initial protoporphy­rin release, the fluorescence intensities from all the porphyrins present in the culture vanished exponentially with the irradiation time. The metal containing form of fluorescent coproporphyrin, with a maximum emission at 580 nm, was photobleached about ten times faster than the free base forms of coproporphyrin and protoporphyrin. Among these three fluorescent substances in the cell culture only the free base forms of the porphyrins have longer lifetimes than the cells themselves irradiated at the same conditions.

Photsynthetic membranes of Rhodopseudomonas virids and Ectothiorhodospira halochloris were treated with proteinase K. The photochemical activity (light minus dark difference spectra) were compared to the polypeptide composition (SDS-polyacrylamide gel analysis). In E. halo­chloris, difference bands appear at 806 (+), 838 (+) and 854 nm (-) . All three decrease in intensity upon incubation with proteinase K., but this decrease is much slower than the proteolysis of both the reaction center and antenna related polypeptides. Photochemical activity remains high as long as a small part of the RC and two lower molecular weight polypeptides M* (22.0 kDa) and B* (15.3 kDa) are present. The M subunit is the most stable polypeptide in the RC of Rp. viridis too, and the photochemical activity is related to the remainder of this and to the one newly formed polypeptide (15.3 kDa), but doesn’t show the typical absorption shift of the antenna (B 800/1020 → B 800/960). The results are discussed quantitatively and compared to those obtained from Bchl α containing organisms.

Pheonlic herbicides, being quite hydrophilic as compared to classical photosystem-II inhibitors do not exhibit a correlation between inhibitory activity and partition coefficient. Sensitivity against phenolic herbicides, however, as observed with atrazine-resistant thylakoids from higher plants mutants (Brassica napus) is increased by higher partition coefficients.

Biosynthesis and turnover of the different cell wall components have been studied during the vegetative cell cycle of Chlamydomonas reinhardii by pulse-labelling with [ 3 H]proline and [ 35 S]methionine and by pulse-chase experiments. Two phases of biosynthesis of insoluble cell wall material could be distinguished: 1. de novo synthesis of the daughter cell walls during cytokinesis and 2. cell wall enlargement during cell growth. During the cell enlargement period, a turnover of the insoluble wall component was observed. The released fragments were found to be accumulated in the culture medium. The LiCl-soluble cell wall glycoproteins were found to be precursors of the insoluble cell wall layer. Biosynthesis of the LiCl-soluble cell wall glycoproteins was observed mainly during the time period between cytokinesis and the end of the following cell enlargement period. Labelling of all the cell wall components was found to be strongly reduced during the time period between the end of the growth phase and cytokinesis. During cytokinesis, labelling of the insoluble cell wall material preceded the incorporation of radioactive precursors into the LiCl-soluble wall fraction.

In analogy to experimental results previously obtained with vimentin and neurofilament triplet proteins, the intermediate filament (IF) proteins desmin and glial fibrillary acidic protein (GFAP) were also found to have high capacities to associate with nucleic acids. Employing a collection of native and heat-denatured pro-and eukaryotic DNAs, a series of naturally occurring single­ stranded (ss)RNAs and a variety of synthetic polynucleotides of the RNA and DNA type, both proteins could be shown to bind preferentially to single-stranded polynucleotides. In the case of ssDNA and synthetic polyribonucleotides, a clear dependency of the binding activity on the G-content of the nucleic acids was demonstrated. The interaction of desmin with ssDNA and tRNA was characterized by strong cooperativity. When a mixture of desmin and vimentin was offered to excess ssDNA, the cooperativity effect brought about segregation of both protein species into two distinct populations of deoxyribonucleoprotein particles with substantially differ­ent sedimentation rates; this segregation is in sharp contrast to the ability of desmin and vimentin to form heteropolymers in filament assembly. In general, desmin and GFAP were found to be similar to vimentin and neurofilament proteins in their nucleic acid-binding properties. However, there were also striking differences between individual non-epithelial IF proteins at this level.

Rat liver chromatin has been fractionated by different solubility in solvents of 155 mᴍ ionic strength in soluble S and insoluble I-chromatin. Histone H1 content is lower in S as compared to I-chromatin. The HMG1/2 nonhistone proteins are observed in S-chromatin and in the nuclear pelleted residue from the chromatin isolation procedure, but no amount can be detected in I-chromatin. Thermal denaturation profiles and CD-spectra are different for S and I-chromatin indicating distinct interactions between DNA and proteins in the chromatin molecules. Both effects, differing protein content and distinct DNA-protein interactions, can be correlated with solubility and insolubility being the result of charge-charge interactions between chromatin molecules and ionic components of the solvent.

Equilibrium dialysis studies and sedimentation experiments with soluble rat liver chromatin and radioactive 45 Ca support the hypothesis that there is a specific binding of Ca 2+ to chromatin molecules. At low ionic strength the binding constant is about 12 1/mᴍ indicating that more than half of the negative charges of the chromatin molecules are neutralized due to the binding of Ca 2+ at appropriate concentrations. This effect possibly accounts for low concentration of Ca 2+ and other divalent cations being effective in inducing compact higher order structures of chromatin. Monovalent cations are not specifically bound, but high concentrations cause unspecific shielding of the charges on the chromatin molecules and thus structural transformation. Even under these high salt conditions a specific binding of Ca 2+ to chromatin occurs. The binding constant is, however, only 0.4 1/mᴍ due to the lower effective concentration of the charged chromatin molecules which is reduced by unspecific neutralizing.

Based on the different absorption spectra of H 2 O and D 2 O in the near infrared (NIR, 800-2200 nm) a strategy is developed to measure H 2O /D 2 O-fluxes in thin sections. The physical background of the method is presented. Results on the H 2 O/D 2 O-fluxes in filter paper and plant leaves together with both the physical and biological limitations of the new method are discussed.

(R)Mandelonitrile and the cyanogenic glucoside prunasin have been established as the source of HCN in all stages of the Australian beetle Paropsis atomaria. Quantitative data for both compounds and all life stages are presented. The larvae contain a β-glucosidase activity capable of hydrolysing prunasin and also β-cyanoalanine synthase activity enabling the disposal of free cyanide.

Comparative studies of circadian activity rhythms were performed with three stocks of the species Pterostichus rhaeticus Heer (from Southern Europe, Central Europe, and the Subarctic) and with one Central European stock of the sibling species Pterostichus nigrita Paykull. The hehaviour was investigated in various LD conditions, constant conditions including LL with changes of the illumination intensity and constant darkness with light pulses. The data were analysed with the program system “Timesdia” containing various techniques like power spectrum estimation, periodogram analysis and complex demodulation. This allowed to reveal the time course of rhythms and to determine the number of phase jumps as a measure for precision. With the help of these methods, the activity patterns are classified in different types of behaviour: synchronization, temporarily synchronization, aperiodic behaviour, free run, temporarily free run, relative entrainment, and relative coordination. Besides graduated differences between the populations in south-to-north direction (e.g. in the tendency to rhythmic behaviour) the analysis revealed splitting of rhythms into two or even three components of different frequencies and transitions from rhythmic to aperiodic behaviour or vice versa after arbitrarily chosen light pulses in constant darkness. This behaviour cannot be explained by a system which is composed of one single limit cycle oscillator, but the results rather support a multi-oscillator model.

Four flavonoids have been identified from the leaf and stem resin produced by Baccharis bigelovii (Asteraceae) in addition to twelve previously described aglycones. A flavonol (alnusin) and a dihydroflavonol (alnustinol) with 3,5,7-trihydroxy substitution had both been reported only twice before. The novel compound 2β,5,7-trihydroxy-flavanone is remarkable for its 2β-substitution at the heterocyclic ring. 7-Benzoyl-chrysin is the first flavone aglycone found as ester and the first flavonoid aglycone at all that is esterified with an aromatic acid.

From the herbage of Passiflora incarnata have been isolated eight flavone-C-glycosides based on apigenin and luteolin, six of them had not been found before in that plant. The constitution of all compounds is prooven.

Several derivatives and analogs of the recently reported antiproliferative and antitumor agent trans-bis(salicylaldoximato) copper(II) (CuSAO 2 ) have been prepared and tested for antiproliferative activity against L1210 leukemia cells in vitro. The salicylaldimine analog of CuSAO 2 had a very strong antiproliferative activity, the 2-day IC 50 value being lower than 3 μg/ml- The 2,3-dihydroxybenzaldoxime analog was equally active with CuSAO 2 , while the corresponding 2,5-dihydroxy derivative had a slightly lower activity. The 2,3,4-trihydroxybenzaldoxime derivative had a much lower activity than had the dihydroxybenzaldoxime derivatives. The zinc(II) analog of CuSAO 2 had only a low antiproliferative activity. The ligand of CuSAO 2 , salicylaldoxime, resembles pyridoxal oxime, a vitamin B 6 antagonist and a powerful inhibitor of pyridoxal kinase. An attempt to reduce the toxicity of CuSAO 2 in vivo with pyridoxal hydrochloride led to increased toxicity.

Serum and tissue copper concentration was determined in mice bearing induced fibrosarcoma after successful chemotherapeutic treatment. The results showed a significant depression in copper concentration in the serum and tumour tissue after treatment with anti-cancer drugs. However, the liver copper concentration showed no significant change in treated groups of mice compared to before treatment.