Band 50, Heft 9-10

The metabolism of [8- 14 C ]adenine and [2- 14 C]caffeine was examined in leaf segments from flush shoots of tea cultivars with high and low caffeine content. The caffeine biosynthesis pathway from AMP via theobromine was operative in both high and low caffeine containing cultivars. There was a m ore rapid rate of caffeine biosynthesis from [8- 14 C ]adenine in the high caffeine cultivars while the rate of degradation of both adenine nucleotides and caffeine into CO 2 was greatest in cultivars with a low endogenous caffeine content. Cell-free p reparations from tea shoots contained an N-methyltransferase, that is a keyenzyme in the caffeine biosynthesis pathway; more in-vitro activity was detected in preparations from high caffeine containing cultivars. The data obtained suggest that the high caffeine containing cultivars have a more rapid rate of caffeine biosynthesis and a slower rate of caffeine catabolism than cultivars with a low endogenous caffeine content

The distribution of hydroxycinnamoylmalic acids (HCM acids) in 25 species and 2 subspecies of Papaveraceae and Fumariaceae representing 14 genera was investigated by HPLC and subsequent diode array detection (DAD). In case of similar chromatographic and UV-spectrometric behavior of HCM acids and other hydroxycinnamic acid derivatives liquid chromatography-thermospray/mass spectrometry (LC-TSP/MS) was used for positive identification. Ten of the species were found to contain caffeoyl-, p-coumaroyl-. and feruloylmalic acid whereas Chelidonium majus only showed the presence of caffeoylmalic acid. Sinapoylmalic acid could not be detected. The quantitative determ ination by HPLC at 330 nm yielded the highest content of caffeoylmalic acid for Fumciria officinalis and F. capreolata, and the lowest for Chelidonium majus. Isolated HCM acids showed positive optical rotation for members of the Papaveraceae and negative values for m em bers of the Fum ariaceae. This points to an esterification of hydroxycinnam ic acids with (-)-ʟ -malic acid for the Papaveraceae and with (+)-ᴅ-malic acid for the Fumariaceae. For plants containing HCM acids the contents of free l - and ᴅ-malic acid w ere determ ined enzym atically Yields of ʟ-malic acid were higher for Papaveraceae than for Fum ariaceae; ᴅ-malic acid was detectable only in traces for all investigated species. The Fum ariaceae showed a ca. 1:1 relationship between free ʟ-malate and esterified ᴅ-malate. This suggests an enzym atic racem ization of the original ʟ-malate and a subsequent esterification of the intermediate ᴅ-malate with hydroxycinnamic acids

From the culture media of Pseudomonas aptata 4b and of Pseudomonas fluorescens ATCC 13525 5,6-dihydropyoverdin-7-sulfonic acids could be isolated. Their possible role in the biogenetic pathway leading to the pyoverdins will be discussed

Pyoverdin type siderophores produced by six fluorescent Pseudomonas strains isolated from different rhizospheres were purified and characterized. The purified ferri-pyoverdins were tested for their ability to promote the growth of other strains grown under iron deficiency conditions. Only the one obtained from Pseudomonas putida BTP1 did not act as a growth promoter. The structure of the BTP1 siderophore was elucidated by spectroscopic methods and degradation studies. It turned out that it contains a chromophore which differs from the one typical for pyoverdins insofar as it carries the carboxyl group in 3- rather than in 1-position ((3S)-5-amino-1,2-dihydro-8,9-dihydroxy-3 H -pyrimido[1,2 a]quinoline-3-carboxylic acid). The amino group of the chromophore is substituted with the 5-carboxyl group of ʟ-glutamic acid and its carboxyl group with the N -terminus of the peptide ʟ -A sp -ʟ -A la-ʟ -Asp - ᴅ-N 5 - Ac - N 5 - OH - Orn - ʟ - Ser - ʟ - ᴄ - N 5 - OH - Orn . This isopyoverdin fits into the biogenetic scheme which postulates ferribactins as the precursors of pyoverdins

Phosphoserine am inotransferase (EC 2.6.1.52) has been purified from Scenedesmus obliquus. mutant C -2A′, as reported previously (Stolz and Dörnemann, 1994). The current studies on its catalytic properties, involving initial reaction velocities as a function of the phosphoserine concentration at various fixed concentrations of 2-oxoglutarate as aminoacceptor, indicate a bi-bi ping pong mechanism . The application of a variety of substrate analogues of phosphoserine revealed no significant metabolisation of these com pounds and thus a considerable specificity of the enzyme. 4,5-dioxovalerate with glutamate as am inodonor is effective as competitive substrate to phosphohydroxypyruvate in the forward reaction and yields 5-am inolevulinate. 4.5-Dioxovalerate and glutam ate-1-semialdehyde can both serve as competitive aminoacceptor in the reverse reaction with phosphoserine and as substrate with 2-oxoglutarate as am inoacceptor. Comparison of the phosphoserine transamination with the transamination of 4,5-dioxovalerate revealed for both reactions a pH -optim um of 6 .8 -7 .0 in Mes/Bis-Trisbuffer. How ever, the K m -values and the V max for phosphoserine and 2-oxoglutarate on the one side, and 4,5-dioxovalerate and glutamate on the other were found to differ by orders of magnitude

A low ʟ-arginine metabolizing enzyme (L-AME) activity leading to ornithine, urea and additional products not identified so far could be detected in photosystem II (PS II) membranes of spinach and of the chlorophyll deficient tobacco mutant Su/su. The detectable L-AME activity was very low in untreated PS II membranes, but increased significantly (about 10 fold) when the extrinsic peptides (psbO, P and Q gene products) were removed - suggesting that the L-AME is exposed at the lumen side of PS II. It was possible to isolate the detergent-solubilized protein from CaCl 2 -washed PS II membranes of spinach by a combination of anion and cation exchange columns. On the basis of SDS PAGE the protein was homogenous and had an apparent molecular mass of 7 kDa. N-terminal sequencing of the polypeptide gave a contiguous sequence of 20 amino acids showing no homologies to PS II polypeptides as yet sequenced. After chromatography of the L-AME on an anion exchange column at pH 9.5 (last purification step) a completely inactive enzyme was obtained. Maximal reactivation was achieved by dialyzing the protein against Hepes-NaOH buffer in the pH range of 6.5 to 7.5 containing 100 mᴍ chloride or sulfate (being the most effective anions). The L-AME activity was totally dependent on manganese added to the reaction mixture. Moreover, there were indications of a second cation binding site being more sequestered and requiring bound Ca 2+ or Mn 2+ for activity (Sr 2+ was less effective and Mg 2+ was ineffective). There are indications that the protein contains a redox active group - possibly an aminoacid- derived quinonoid (based on a redox cycling assay with glycine and nitroblue tetrazolium). The capability of this PS II associated protein to bind the cofactors of water oxidation and having a redox active group (preliminary results) suggests that this protein might be functional in photosynthetic water oxidation. This is further supported by the fact that the isolated L-AME has a low catalase activity

High resolution melting profiles of four linearised plasmids (pUC9, pGV403, pHP2, and pBR322) were recorded by means of UV absorbance vs. teperature scaning. The set of transitions obtained for each plasmid are compared to each another and to the transitions obtained for their excised particular antibiotica resistance gene. It can be shown that each gene leaves a charactersitic mark on the melting profile of its parental plasmid

Two homologues of cereal grain resorcinolic lipids, 5-n-heptadecylresorcinol and 5-n-hepta-decenylresorcinol studied in the system employing yeast cell protoplasts showed marked fusiogenic activity. The frequency of hybrid formation induced by studied amphiphiles was significantly higher than that obtained with the use of 40% (w/v) polyethylene glycol 4000. The resorcinolic lipids as fusion-inducing agents did no affect regeneration of the cellular wall. The fusiogenic activity of resorcinolic lipids lost when calcium ions were absent in the medium. Fusiogenic activity of studied amphiphiles is related to their ability to induce non­ bilayer structures within the cellular membranes.

Paraoxon. the main active metabolite of the organophosphorus insecticide parathion. exerted a dose-dependent inhibitory effect on the activity of pig kidney (Na + + K + )-ATPase contained in microsomal fraction and purified from it. Substrate kinetics studies revealed the existence of two active sites with high and low affinity to ATP. The Dixon analysis of the mode of the inhibition indicated its noncompetitive character. The purified enzyme was more affected than enzyme contained in the microsomal fraction. The inhibition constant K, ranged from 73 to 246 μm depending on the type of preparation. The Hill coefficient (n) fulfilled the relationship 1<n<3. These properties of the interaction suggest the cooperative binding of paraoxon to the enzyme. An indirect mechanism of the interaction was proposed: paraoxon could inhibit the activity of the (N a + + K + )-ATPase by excluding the enzyme protein from its normal lipid milieu

Esterases in nine rat hepatic and hepatoma-derived cell lines and normal rat liver homogenate were detected by polyacrylamide gel electrophoresis coupled with active staining with a-naphthyl acetate or butyrate as a substrate. The esterase band patterns of the non-cancerous and oncogene-transformed cell lines were alike, but different from those of hepatoma cell lines and normal rat liver homogenate. The former groups of cells might have completely lost the characteristics of rat liver parenchymal cells, or else they might have their origin at cells other than liver parenchyma. The esterase patterns of the hepatoma cell lines (e.g., McA-RH7777) and the normal rat liver highly resembled with each other, exemplifying the slight biochemical deviation of cancer from normal cells. However, two-dimensional electrophoretogram for the McA-RH7777 cell line showed a prominent esterase spot {p/ 6.0-M r 110 kDa) that was lacking in the normal liver. This result indicates that there is invariably some change in esterase expression between the cancer cells and the normal liver cells

Analysis of the tropical marine sponge Axinella carteri afforded six unusual alkaloids, including the new brominated guanidine derivative 3-bromo-hymenialdisine. The structure elucidation of the new alkaloid is described. The alkaloid patterns of sponges collected in Indonesia or in the Philippines were shown to be qualitatively identical suggesting de novo synthesis by the sponge or by endosymbiontic microorganisms rather than uptake by filterfeeding. All alkaloids were screened for insecticidal activity as well as for cytotoxicity. The guanidine alkaloids hymenialdisine and debromohymenialdisine exhibited insecticidal activity towards neonate larvae of the polyphagous pest insect Spodoptera littoralis (LD 50 s of 88 and 125 ppm, respectively), when incorporated into artificial diet and offered to the larvae in a chronic feeding bioassay. The remaining alkaloids, including the new compound, were inactive in this bioassay. Cytotoxicity was studied in vitro using L5178y mouse lymphoma cells. Debromohymenialdisine was again the most active compound (ED 50 1.8 μg/ml) followed by hymenialdisine and 3-bromohymenialdisine, which were essentially equitoxic and exhibited ED 50 s of 3.9 μg/ml in both cases. The remaining alkaloids were inactive against this cell line

Both sexes of the dung beetle Pachylomerus femoralis were observed to be attracted to the male dung beetle Kheper lamarcki when it exhibited typical calling behaviour including the release of a white flocculent sex-attracting secretion. Using GC-MS analysis and gas chromatography with electroantennographic (E A D ) and flame ionization detection (FID) in parallel, methyl and ethyl propanoate. methyl and ethyl butanoate, methyl and ethyl pentanoate, and methyl and ethyl 4-pentenoate were identified as the constituents of the secretion that elicited electroantennographic responses in the antennae of male and female P. femoralis. In field tests, mixtures of these compounds were found to be about as attractive as horse dung. These esters appear to constitute an attractant for P. femoralis, enabling this species which does not form balls, to detect and utilize dung balls formed by K. lamarcki. P. femoralis has been observed to exploit food types as diverse as dung from rhinoceros and primates. It is therefore, possible that, on the other hand, this species utilizes different kairomones to detect various types of dung and that the coincidental presence of some or all of the components of one of these kairomones in the abdominal secretion of K. lamarcki is responsible for its attraction to calling K. lamarcki males

Using techniques that were developed for the concentration and analysis of airborne or­ganic volatiles, methyl salicylate and 1,4-benzoquinone were found to be the only two com ­ pounds present in detectable quantities in the effluvium of Oniticellus egregius. These two compounds, previously identified in the defensive secretions of other arthropods, are pre­sumed to be produced as a defense mechanism against predators, such as birds. Methyl salicylate and 1,4-benzoquinone have thus far not been found to occur in other Scarabaeinae.

During arousal from estivation in land snails. Otala lactea, active metabolic functions are restored within minutes and oxygen consumption increases dramatically. During the transition from the hypoxic conditions of estivation to normoxia it is possible that xanthine oxidase (XO ) in hepatopancreas contributes to the observed lipid peroxidation. Using a fluorometric assay that is based on the oxidation of pterin, the activities and some properties of XO and XO+XDH (sum of XO and xanthine dehydrogenase activities) were measured in hepatopancreas extracts. Km values for pterin for XO and X O +XDH were 9 and 6 μm, respectively, and the Km of XDH for methylene blue was 5 μm . Both XO +XDH and XO activities were inhibited by allopurinol (I 50 = 2 μm ) , pre-incubation at 40 °C, and by 5 min H 2 O 2 pre-exposure. Inclusion of azide in the reaction promoted a rise of approximately 70-fold in the inactivation power of H 2 O 2 due to inhibition of high endogenous catalase activity. The /so for H 2 O 2 of XO +XDH and XO activities in the presence of azide was 0.04 and 0.11 mM , respectively. Unlike the situation for mammalian XO. a previous reduction of O. lactea XO (by pterin) was not necessary to make the enzyme susceptible to H 2 O 2 effects. Interestingly, methylene blue partially prevented both heat- and H20 2-induced inactivation of XO +XDH activity. These data indicate that the formation of an enzyme-methylene blue complex induces protection against heat and oxidative damage at the FAD-active site. Both XO and XO +XDH activities were significantly higher in snails after 35 days of estivation compared with active snails 24 h after arousal from dormancy. The ratio of XO /(XO +XDH) activities was also slightly increased in estivating O. lactea (from 0.07 to 0.09; P < 0.025). XO activity was 0.03 nmol · min -1 · mg protein -1 in estivating snails. Compared with hepatopancreas catalase, XO activity is probably too low to contribute significantly to the net generation of oxyradicals, and hence to peroxidative damage. Rather, the low potential of XO to induce oxidative stress may constitute an adaptive advantage for O. lactea during arousal periods

In photoreceptor cells of invertebrates light triggers an enzyme cascade in which the phos-phoinositide pathway is crucially involved. Likewise, there is growing evidence of an impor­ tant role of cyclic nucleotides, too. To localize these enzymes able to catalyze the formation of cGM P and cAMP, the spatial distribution of guanylate cyclase (EC 4.6.1.2) and adenylate cyclase (EC 4.6.1.1) was determined in photoreceptor cells of the fly. In photoreceptor cells of the blowfly (Calliphora erythrocephala), the electron dense reaction product of guanylate cyclase was found within the phototransducing region, the rhabdomeral microvilli and in the mitochondria. Staining was also observed throughout the cytoplasm of the microvilli. With the same cytochemical method, reaction product for adenylate cyclase was found on the tips of the photosensory membrane, and not in the cytoplasm of the rhabdomeral microvilli. The results presented here further argue for an important role of one or possibly two cyclic nucleotides in the photoreceptor cells, and possibly in the process of phototransduction of in­ vertebrates.

Increased blood plasma concentrations of the sulphur amino acid homocysteine (“homocysteinemia”) have been brought into context with neurodegenerative and arteriosclerotic symptoms and diseases. We recently reported on biochemical model reactions on the prooxidative activity of hom ocysteine including the desactivation of Na + /K + -ATPases and hemolysis of erythrocytes (Preibisch et al., 1993). In this communication we extend our model reactions including the oxidation of methionine, metabolization of pyridoxalphosphate and dihydroxyphenylalanine, desactivations of transaminases and peroxidation of low density lipoprotein

We report here a molecular modeling study of selected conformationally constrained phenylcarbamate local anesthetics in relation to the available pharmacological data that enabled us to develop a receptor-interaction model for this class of drugs. The validity of the model was confirmed on other semirigid analogues prepared for this study. The results suggest that the phenyl ring is most likely involved in a stacking interaction with a complementary receptor site and the tertiary ammonium group is capable of both hydrogen bonding and lipophilic interactions.

6-Hydroxy-dopamine is easily oxidized in aqueous solutions within a pH range of 7-14. Depending on this parameter and the starting concentration of this catechol at least seven different paramagnetic species can be detected. Cyclization to two different indole derivatives of aminochrome type could be demonstrated and the mechanism of formation is discussed. The indoloquinones can easily be reduced to the corresponding semiquinone by an excess of 6-hydroxy-dopamine. All radicals observed react with ascorbic acid in a redox reaction, forming the corresponding catechol and the ascorbic acid semidione

Hypochlorous Acid, Cartilage, Chondroitinsulphate, Carbon NM R Spectroscopy It is a well known fact that neutrophil-derived hypochlorous acid plays an important role in cartilage destruction during rheumatoid arthritis. It has been shown by 1 H NMR spectro­scopy in a previous paper (Schiller et al. (1994), Biol. Chem. Hoppe-Seyler 375, 167-172) that sodium hypochlorite affects primarily the N-acetyl side chains of polymeric carbohydrates of cartilage like chondroitinsulphate and hyaluronic acid. An instable intermediate, likely to be a chloramine, is involved in these processes. The present paper deals with the application of carbon NMR spectroscopy for the study of these degradation processes, because carbon NMR gives the opportunity to detect changes on the single sugar ring carbons. Although it was not possible to prove the involvement of an intermediate, because of its fast hydrolysis, we were able to show that the reaction between sodium hypochlorite and N-acetylglucos-amine affects mainly the side chain, accompanied by of a large excess of sodium hypochlorite leads to under the formation of formiate.

An angular-type dihydropyranocoumaringlucoside, namely, cis-Khellactone-3′-β-D-glucopyranoside (1) was isolated from the fruits of Ammi visnaga (L.) LAM. (Apiaceae). The structure was established on the basis of acid hydrolysis and spectral data

The inhibitory effect of benzoic acid hydrazide (bah) and 3-methoxybenzoic acid hydrazide (mbah) on Saccharomyces cerevisiae strains has been compared to that of their platinum(II) complexes: cis-[Pt(bah) 2 X 2 ], cis- [Pt(NH 3 )(bah)Cl 2 ]-0.5 H 2 O. cis-[Pt(mbah) 2 X 2 ] and cis- [Pt(NH 3 )(mbah)Cl 2 ] (X = Cl, Br = I), and cis- [Pt(NH 3 ) 2 Cl 2 ]. The minimal inhibitory concentrations for bah and mbah were 5000-20.000 μm whereas for their Pt(II) complexes they were much less (25-800 jam) and did not exceed these of cisplatin (100-800 μm ) . The activity of the Pt(II) complexes of bah and mbah varied in wide ranges for the different yeast strains tested. Osmotically unstable mutants were found to be more susceptible. The most active complexes were [Pt(NH 3 )(bah)Cl 2 ] · 0.5 H 2 O and [Pt(NH 3 )(mbah)Cl 2

Two multiple antigen peptides consisting of 6 and 7 amino acid residues, respectively, plus a 12-residue fragment, used as a control, all linked to a polylysine core, were used as immunogens in rabbits in order to obtain an immune response. Rabbit antisera against such polymers were then tested in ELISA against a panel of antigens in order to analyze the specificites of the resulting antibodies. The responses were different for all three immunogens, being partially or totally directed, for two of the three compounds, including the 12-residue control MAP peptide, against the polylysyl core, which is considered as non immunogenic. The third MAP polymer was practically unable to elicit an immune response

We have shown that the distribution of lengths of site nucleated microtubules obey an algebraic power law relationship D(s) = As -τ , where D(s) is relative number of microtubules with length 5, A and τ are constants. This relationship indicates the possibility of a self-organized criticality in the dynamic instability of microtubule growth