Volume 44, Issue 7-8

Amino Acid Conjugates The conform ation of the side chain of indol-3-yl-acetic acid conjugates with am ino acids (L-alanine, glycine, L-i-leucine, L-a-aminobutyric acid, DL-aspartic acid, DL-phenylalanine, and 6-am inovaleric acid) has been studied by 1 H -NMR -NOE -difference spectroscopy. Com parison with the conform ation in the solid state is given in the light of structure-activity relationships.

A polar fraction obtained from banana fruit pulp by high-vacuum distillation / solvent extraction (pentane-dichlorom ethane, 2 + 1) and subsequent liquid chromatography on silica gel was derivatized with isopropylisocyanate. Separation of enantiom ers of heptan-2-ol and Z-4-hepten-2-ol was performed via their carbamates by means of multidimensional gas chromatography using an achiral (D B -5, column 1) and a chiral (X E -60, column 2) capillary column. The enantiomeric ratios of heptan-2-ol and Z-4-hepten-2-ol were determ ined to be (/?)( - ) 39% and (S)( + ) 61% as well as (/? )(+ ) 26% and (S )(-) 74% , respectively. The absolute configuration of optically enriched ( 5 ) (-)-Z-4-hepten-2-ol was determined from its (R)-2-phenylpropionic acid ester by 1 H NM R spectroscopy.

From gam etophytes of Philonotis fontana six biflavonoids have been isolated. Two of them - philonotisflavone and 2,3-dihydrophilonotisflavone - were hitherto unknow; their structures have been elucidated spectroscopically.

The epicuticular wax of Euphorbia cyparissias contains pentacyclic triterpenoids (34%) and primary alcohols (31%) as major com ponents. The major triterpenoids are triterpenols (23%) consisting of α-and β-amyrin, glutinol, 24-m ethylenecycloartenol and ψ-taraxasterol. The triter­ penones (10.5%) are com posed of taraxerone. a -and β-amyrinone, lupenone, glutinone, ψ-taraxasterone, β-fernenone and three further unidentified triterpenes. α-Amyrin was found esterified with hom ologous series of fatty acids. Minor components are alkanes, wax esters, aldehydes and free fatty acids.

The existence of sympatric interspecific hybrids of Sideritis serrata Cav. ex Lag. and S. bou r­ gaeana Boiss. Reut, has been shown by HPLC analysis of the flavonoid glycosides accumulated in the cell sap, and by the methylated flavonoid aglycones exudated by the plants and deposited onto the plant surfaces. The hybrids tend to produce in a single plant the compounds characteristic of the parental taxa. This “complementation” effect in the hybrids permitted relatively easy detec­tion of F1 hybrids. Generally, there was a close correlation between the biochemical results and a morphological analysis which used the Anderson’s index, although a specimen which morphologi­cally was a hybrid, showed a flavonoid pattern as S. bourgaeana. The external flavonoids analysis has revealed that the activity of sideritoflavone O -methyltransferases is much more important in S. serrata than in S. bourgaeana, while the hybrids show intermediate values. M oreover, S. bour­gaeana accumulates sideritoflavone 3′-m ethylether whereas S. serrata accum ulates the 4′-methylether. The hybrids produce both com pounds in similar amounts. In this study, external flavonoids have proved more useful than vacuolar flavonoid glycosides in the biochemical documentation of hybridization. This is probably due to the fact that methylated flavonoid aglycones are physiologi­cally more stable as they are externally located and therefore preserved from the action of enzymes.

Using Coreopsis tinctoria liquid cultures of Agrobacterium rhizogenes transformed roots (socalled “hairy roots”) and non-transformed roots have been established. The production of different phenylpropanoid com pounds by these cultures grown in light or darkness, respectively, was compared. Whereas the com position of phenylpropanoids in the root cultures was not influenced by the culture conditions, the total production of phenylpropanoids as well as the formation of single com pounds each analyzed were higher in darkness than in light. The biomass production of the transformed root cultures exceeded that of non-transformed root cultures by a factor of 2.6. A t the same time com pound accumulation paralleled the biomass production, so that the dark grown transformed root cultures were producing maximum yields of several phenylpropanoids, especially l′-isobutyryloxy-eugenol-isobutyrate (7 mg per culture flask during a single culture passage).

A highly efficient method employing NaSCN as a chaotropic agent was used to dissociate the membrane-bound porphobilinogen-deaminase. The same sequence of steps was applied for purifying both soluble and membrane dissociated porphobilinogen-deam inase. The chromatographic behaviour of both proteins was quite similar. Euglena gracilis deam inase appears to exist in an equilibrium mixture of two active species of relative molecular masses of 40000 and 20000.

A new continuous fluorimetric assay for AMP deaminase activity is described. The m ethod makes use of a fluorescent analog of 5′-AMP, formycin-5′-phosphate (5′-FM P), which undergoes deam ination to formycin B-5′-phosphate, not fluorescent at neutral pH. The pH -dependence for deamination of 5′-FMP is similar to that for 5′-AMP, but shifted about 0.2 units to more acidic pH. Deamination of 5′-FM P may also be followed spectrophotometrically at 306 nm, permitting better assays of crude extracts. Some kinetic results obtained by means of the new method for AMP deaminase from chick and rabbit skeletal muscle are presented. In particular it was found that the natural product of deamination, 5′-IMP exhibited allosteric inhibition of the chick enzyme with K, values 1.6mM , 1.2mM and 1.0mM at pH 5.8, 6.5 and 7.3, respectively. Activation by diadenosine tetraphosphate, Ap 4 A , reported for mouse muscle AMP deaminase, has not been noted for the chick enzyme. Inhibition by the transition state analogs, coformycin and 2′-deoxycoformycin, was observed for both rabbit and chick deaminases with K i values ~ 1 μM and ~ 1.6 μM respectively. Kinetic data for coformycin-5′-phosphate show it to be a tight-binding inhibitor with K, < 0.6 × 10 -9 M as compared to 1 × 10 -9 m for 2′-deoxycoformycin-5′-phosphate.

Quantitative analysis of nine major com ponents of the head extracts of workers and queens of Apis mellifera intermissa was carried out. Q ueen and worker component patterns were distinct. The patterns of both castes changed with age, and the worker patterns were also affected by being reared in the absence of a queen. The genetic variance of the component composition of the extracts was found to be higher in the queens than the workers. This may have functional significance in the recognition by workers of their own queen.

The total lipids of the commercial land snail Eobania vermiculata (Gastropoda, Pulm onata, Stylom m atophora) are found to constitute a small percentage (0.8% ) of the wet tissue, which is comparable to that reported for other gastropods. Polar lipid components comprise 61.4% of the total lipids. The individual lipid classes obtained by column chromatographic fractionation were purified by preparative TLC or by column chromatography and their structure was confirmed by a combination of chromatographic and analytical determinations before and after mild alkaline hydrolysis and/or (dry) acid methanolysis and by IR analysis. Neutral lipids represent 36.4% of total lipids, containing cholesterol, cholesterol esters and triglycerides as their major components (26.2% , 29.1% and 25.5% respectively). They contain also a significant amount (14%) of free glyceryl ethers, which are found in a mollusc for the first time. The overall composition of the polar lipids (mol/100 mol lipid-P) was found as follows: Cardiolipin, 2.9; phosphatidylethanolamine, 24.9 (of which 19.8% plasmalogen analog); phosphatidylcholine, 49.2 (of which 45.6% glycerylether analog); ceramide aminoethylphosphonate, 7.5 plus 0.01 (another three minor species); diglyceride-am noethylphosphonate, 6.3; Sphingoethanolamine 1.65 (for the first time found and structurally studied in a land gastropod); and phosphatidic acid 1.1. Unsaturated fatty acyl groups represent about 72.6 and 44.1 respectively in phosphatidylethanolamine and phosphatidylcholine. A significant amount (70.5% ) of unsaturated fatty acids is concentrated in neutral lipids. The C 16:0 alk-1-enyl chain was found to predominate (55.6% ) in the side chains of ethanolamine plasmalogen. Batyl alcohol was found as the main glycerylether bound to choline phosphate (97.5% ). Saturated fatty acyl groups with 16 carbon atoms were main components (54%) of the major ceramide aminoethylphosphonate species.

Many derivatives of 2,3-dim ethoxy-4-hydroxypyridine, which were designed from examination of the structure-activity relationship of piericidins, were tested for inhibition of NADH-UQ reductase. The lipophilic side chain of those compounds was indicated to be a key part for activity and its optimal length was conjectured. By the use of two different phases of assay material, intact mitochondria and submitochondria, the size of a membrane effect was shown to depend on the structure of the side chain. 4-Hydroxyquinoline derivatives were also tested for an analogous role in relation to the electron transport function of menaquinone, and they were proven to be inhibitors of NADH-UQ reductase as good as the pyridine derivatives.

The purple sulfur bacterium T. roseopersicina is able to grow chemoautotrophically in the dark under micro- to semiaerobic conditions. Under the latter cell yield and growth rate are considerably reduced. During chemo- and phototrophic growth reduced sulfur compounds are metabolized by the same pathway, i.e. oxidized to sulfate via adenylylsulfate. APS reductase (EC 1.8.99.2), ADP sulfurylase (EC 2.7.7.5) and ATP sulfurylase (EC 2.7.7.4) could be shown in all the strains tested, whereas only strain BBS contained an AMP independent sulfite-oxidizing activity under photo- as well as under chemotrophic conditions. Not only ADP but also ATP sulfurylase perform a dissimilatory function proven by their high specific activities. In contrast to the enzyme of T. roseopersicina strain 6311 APS reductases from strains M1 and BBS are strictly membrane-bound. The enzyme from strain M1 was solubilized, enriched and characterized. While the K M values of purified APS reductase remain unaffected, specific activities of APS reductase, ATP and ADP sulfurylase are increased substantially under chemotrophic growth conditions.

The conversion of nicotinic acid to N-conjugates by either m ethylation or glucosylation has been investigated in leaves and tubers from potato plants, in potato callus and suspension cultures and in plants regenerated from such cultures. While nicotinic acid-N-glucosyltransferase activity could be detected in the cells and tissues at each level of differentiation, the N-methyltransferase (trigonelline formation) was only found in organized leaf tissue.

Particulate (15,000 x g ) fractions from developing seeds of mustard (Sinapis alba), honesty (Lunaria annua), and nasturtium (Tropaeolum majus) synthesize radioactive very long chain (n9)-(Z)-monounsaturated fatty acids, e.g. gadoleic (20:1). erucic (22:1). and nervonic (24:1) acids from [1- 14 C ]oleoyl-CoA with malonyl-CoA or from [2- 14 C]m alonyl-CoA with oleoyl-CoA . Pyridine nucleotides are required for the elongation reactions. Both NADH and NADPH are equally effective for the formation of 20:1, but NADPH is the preferred reductant for the formation of 22:1, which suggest the existence of separate elongase systems. Detergents, such as Triton X-100 and octyl thioglucoside, stimulate the elongation at low concentrations, but they are inhibitory at high concentrations. Partial solubilization of the elongase system using these detergents has been achieved.

The am ounts of free, conjugated (hydroxycinnamic acid amides) and insoluble (poly)amines (putrescine, spermidine, spermine) as well as hydroxycinnamic acid esters (quinic acid esters) were determined during development of male inflorescences of Corylus avellana L. The location of these compounds either in sporophytic or gametophytic tissues and the possible correlation of their accumulation with exine formation are discussed.

The classification of methanogenic bacteria as archaebacteria based on 16 s rRNA sequence analysis is currently in dispute. To provide an alternative molecular marker, the polymerase chain reaction technique was used to amplify a 930 bp fragment of Methanococcus thermolithotrophicus genomic DNA corresponding to the catalytic domain of the membrane H + -ATPase. The deduced amino acid sequence was 54-58% identical to the ~ 70 kDa subunits of Sulfolobus acidocaldarius and the eukaryotic vacuolar-type H + -A T P ase, and only 29% identical to the beta subunit of the eubacterial-type F 0 F 1 -ATPases. Interestingly, a highly conserved aspartate residue in the phosphorylation domain of E 1 E 2-A TPases (P-type) is conserved in the Methanococcus sequence, but is absent from all other known vacuolar and F 0 F 1 -ATPases. This suggests that the H + -ATPase of M. thermolithotrophicus, like that of M. voltae, may have a phosphorylated intermediate, despite belonging to the vacuolar-type class of proton pumps. Phylogenetic analysis using Felsenstein’s maximum likelihood method and Lake’s evolutionary parsimony method confirmed that the H + - ATPases of the two archaebacteria, Methanococcus and Sulfolobus, when compared to eukaryotic vacuolar-type ATPases and eubacterial F 0 F 1- ATPases, form a monophyletic group.

1. The in vivo effect of estradiol and domperidone (a hypophysis stimulator of prolactin secretion) in immature ovariectomized-adrenalectomized and hypophysectomized rat mammary gland was studied. 2. γ-Glutamyltranspeptidase activity was used to evaluate the role of estradiol in the specific response of the gland to prolactin. 3. Our results suggest that the activity of γ-glutamyltranspeptidase, like casein and lactose synthetase, is part of the specific response of the gland to prolactin.

Numerous studies have demonstrated induction of proteins in plant tissues by wounding and infestations by various pests and pathogens. Lipoxygenase (LOX) is among the proteins that has been found to be induced by pathogens, but detailed information on the induction of LOX has not been reported. We have found a large (up to 10-fold) increase in LOX activity upon wounding of soybean (Glycine max L. Merr.) leaves and variable increases due to feeding of the twospotted spider mite. This induction in LOX activity was reflected in increases in amounts of both LOX protein and transcripts suggesting that the induction was at the transcriptional level. LOX activity was also found to be increased in unwounded leaves from plants with wounded leaves lower on the stem indicating that translocatable factors can cause remote induction of LOX activity.

A crude cell-free extract from cultures of Fusarium sp. strain C37410-90 possessed significant esterase activity and hydrolyzed the trichothecene mycotoxin 3-acetyldeoxynivalenol (3-AcDON) to deoxynivalenol (DON) in high yield. Smaller amounts of 15-acetyl- and 3,15-diacetyl-esters of DON were also formed. The extract was capable of hydrolyzing a range of natural and semisynthetic trichothecene esters, and showed a high degree of regioselectivity towards position 3.3,4,15-Triacetylscirpentriol (TAS) was efficiently hydrolyzed to 4,5-diacetoxyscirpenol (DAS), and no further transformation was observed. The enzyme activity was partially purified.

It is demonstrated that - similar to chloroplasts - etioplasts isolated from leaves of pea (Pisumsativum L.) and oat seedlings (Avena sativa L.) also possess a high capacity for de novo fatty acid biosynthesis starting from [1- 14 C]acetate. The etioplast system proved to be a suitable test system for inhibitors of de novo fatty acid biosynthesis such as cycloxydim , sethoxydim and diclofop. In contrast to the chloroplast test system , the etioplast system is independent of light and also permits screening of photosynthetic herbicides as potential inhibitors of fatty acid biosynthesis.

Interactions of novobiocin (NB) and/or nalidixic acid (NA) with some cytotoxic agents - UV light (UV), X-rays, methylmethanesulfonate (MMS), bleomycin (BM), adriamycin (AM), cis-di-aminedichloroplatinum (II) (CDDP), mitomycin C (M IT), ethidiumbromide (EB), and suramin (SA) - have been investigated in thymic (T) and splenic (S) cells of the rat in vitro by measuring semiconservative (SDS) and unscheduled (UDS) DNA synthesis as well as sedimentation and viscosity of nucleoids. - Combining NB (900μg/ml) and/or NA (1800-3600 [μg/ml) with UV, MMS, BM, AM, M IT, and SA resulted in additive effects on SDS and UDS. Synergistic actions were observed in T- and S-cells simultaneously treated by NB and CDDP, whereas the effects of NB could be antagonized to some extent by EB and vice versa. In X-irradiated (≥ 28 Gy) cells pretreated by NB (NA), UDS was diminished (T-cells) or enhanced (S-cells). - The results are consistent with the following postulates: 1° in S-cells, DNA is much more supercoiled than in T-cells but in the opposite sense (positive superhelicity). 2° DNA supercoiling (DNA compactness) is influenced therefore by DNA-topoisom erase inhibitors and intercalating agents in a highly agent- and cell-specific manner.

(±)-Aeroplysinin-1, an optically active 1.2-dihydroarene-1.2-diol. was isolated from the marine sponges Verongia aerophoba (+-isomer) and lanthella ardis (--isomer). For the experiments presented we used the +-isomer from Verongia aerophoba. Here we describe the hitherto unknown biological and pharmacological property of this compound to display pronounced anticancer activity against L5178y mouse lymphoma cells (ED 50 : 0.5 μm). Friend erythroleukemia cells (ED 50 : 0.7μm) , human mamma carcinoma cells (ED 50 : 0.3μm) and human colon carcinoma cells (ED 50 : 3.0 μm) in vitro. Furthermore, aeroplysinin caused a preferential inhibition of [ 3 H]thymidine (dThd) incorporation rates in L5178y mouse lymphoma cells if compared with murine spleen lymphocytes in vitro. At concentrations between 1.1 and 28.5 μm, the [ 3 H]dThd incorporation rates in L5178y cells were suppressed to 28% -0% but only to 78% -18% in murine spleen lymphocytes. The same differential effect in vitro was found with the following epithelial cells: 14.70 μm of the compound were required to inhibit normal human fibroblasts to 50% , but only 2.9 μm in the assays with human malign keratinocytes or malignant melanoma cells to observe the same inhibitory effect. Moreover, aeroplysinin-1 displayed antileukemic activity in vivo using the L5178y cell/NMRI mouse system; administered at a dose of 50 mg/kg for five consecutive days, the T/C (% ) value was determined to be 338. Preliminary toxicology studies revealed an acute LD 50 of 202 mg/kg and a subacute LD 50 of 150 mg/kg. Aeroplysinin-1 is neither a direct mutagen nor a premutagen in the umu/Salmonella typhimurium test system.

We have compared by means of rocket immunoelectrophoresis the amount of CF 1 -complex in N. tabacum mutants with that in the wild type N. tabacum var. John William’s Broadleaf. Some of these mutants differ from the wild type not only by higher photosynthetic and photorespiratory activities but also by their chloroplast structure. The amount of CF 1 , present was related to the chlorophyll content. However, no correlation between the amount of CF 1 , present and a higher photosynthetic and photorespiratory activity was found. On the other hand the molar ratio of CF 1 , to chlorophyll seems to depend in these tobacco mutants on the morphological structure of the chloroplasts. In chloroplasts of green phenotypes, in particular in those of the mutants N. tabacum var. Consolation, var. NC 95 and var. Xanthi with high grana stacking as well as in those of the wild type N. tabacum var. JWB one CF 1 -complex occurs per 470-770 chlorophyll molecules. Whereas in chloroplasts of the yellow-green phenotypes with relatively more stroma thylakoids the amount of CF 1 seems to be increased only by a factor of 3-4. The yellow phenotypes contain on the average 1 CF 1 -complex per 60 chlorophyll molecules. The CF 1 -complex in the investigated chlorophyll-deficient tobacco mutants is immunochemically identical to that of the wild type N. tabacum var. John William’s Broadleaf as demonstrated by means of the tandem-crossed immunoelectrophoresis. However, between the CF 1 -complex of tobacco and that of other higher plants such as spinach Spinacia oleracea, only partial identity is observed. Comparative quantitative determinations have shown that one CF 1 -complex binds on the average 8-9 CF 1 -antibodies which shows that the CF 1 -complex of the ATPase is fully exposed in the thylakoid membrane, offering a large adsorption surface to the antibodies. Furthermore, it appears that in the immediate vicinity of the CF 1 -complex large amounts of monogalactolipids are located. Thus, we have calculated that in the variegated NC 95 mutant, chloroplasts of the yellow leaf areas, in which only stroma thylakoids occur, exhibiting only photosystem I activity, as well as in chloroplasts of green leaf areas with a normal ratio of grana and intergrana thylakoids, the molar ratio of monogalactolipids to CF 1 -complex is the same. It looks as if the functionality and activity of the coupling factor of photophosphorylation depended on a certain quantity of monogalactolipid molecules.

Both inorganic orthophosphate and ATP stabilize actin filaments. This is reflected by a reduced nucleotide exchange and by a protection against filament breakdown by SDS or KI. When the filament-stabilizing effect of ATP was maximal, only about 15% of the actin subunits of the filament had bound one molecule of the nucleotide offered in the stabilizing solution.

Ultrastructure of the epicuticular wax of four commercially grown Canadian cultivars of canola (Brassica campestris cvs. Tobin and Candle, B. napus cvs. Altex and Westar) was investigated. Freeze-drying and air-drying methods of specimen preparation for scanning electron microscopy were compared. The freeze-drying method resulted in disruption and possible washing away of wax crystals, whereas there was no visible damage with the air-drying method. The freeze-drying method provided evidence for an amorphous layer of wax beneath the wax crystals. Ultrastructure of wax in the four cultivars was generally similar. All cultivars had an evenly distributed layer of wax crystals superimposed on an amorphous layer of wax. Some trends such as density of wax on leaves and siliques appeared to be species-specific, whereas density of wax on stems did not. There appeared to be at least three types of wax crystals present. These included plate-like, filamentous, and rod-like crystals. The rods were present singly or in blocks.

Cell suspension cultures of Ailanthus altissima (Simaroubaceae) were cultivated under illumination and in the dark. Under influence of a yeast elicitor and cell wall preparations of various Phytophthora species, respectively, alkaloid formation was stimulated. Canthin-6-one and 1- methoxycanthin-6-one accumulation could be increased up to a 125-fold and 2.5-fold, respectively within 96 h by elicitors in dark grown cultures. In response to elicitors canthin-6-one is accumulated in the cells and the growth medium.

An iron-sensitive mutant of E. coli with a Mudl phage insertion in the nrdB gene lacks subunit B2 of the key enzyme of DNA synthesis, ribonucleotide reductase. Nevertheless, these cells are capable of growing in minimal media under anaerobic conditions, indicating a second enzyme or pathway for deoxyribonucleotide synthesis. We here show that ribonucleotide reduction cannot be unambiguously measured in bacterial extracts whereas phosphorylase- catalyzed deoxyribosyl transfer does occur; however these salvage reactions could not function in vivo in the absence of deoxyribosides. It is suggested that the cells possess a specific, anaerobic ribonucleotide reductase which escapes detection under aerobic standard conditions, similar to the situation found in strictly anaerobic methanogens.