Band 47, Heft 5-6

Protein-reactive chemicals and drugs (haptens), such as dinitro- (DNP) or trinitro-phenylated (TNP) reagents or penicillins, are potent inducers of allergic reactions in experimental animal systems as well as in humans. Hapten-specific T lymphocytes are mediators of these disorders. Like other antigens, hapten determinants on target cells are recognized by the T cell’s antigen-specific receptors exclusively in association with gene products of the major histocompatibility complex (MHC). Until recently, the nature of the complex hapten/MHC determinants remained unknown. Atypically, this fact, rather than spurring scientific investigations, caused a rapid loss of interest in haptens as T cell antigens by the majority of immunologists. This situation may now be changing, since we have recently identified MHC-binding, haptenmodified peptides which mediate cellular recognition by T cells via their antigen-specific receptors. Synthetic hapten-peptide conjugates open up new routes in studying the molecular details of hapten recognition by T cells. They may also contribute to a better understanding of what defines an antigen as an allergy-inducing “allergen”.

Bud and leaf exudates from Populus alba, P. balsamifera, P. nigra and P. tremuloides were analyzed by gas chromatography/mass spectrometry and their components were identified. For three of the four species, P. alba, P. balsamifera and P. nigra, the bud and leaf exudates were similar within each species, though there were marked differences between species. Bud and leaf exudates of P. alba contained only hydrocarbons, those of P. balsamifera contained primarily dihydrochalcones and those of P. nigra contained primarily caffeic acid esters. Bud and leaf exudates of P. tremuloides were different both from each other and from those of the other three species. Bud exudate of P. tremuloides contained a range of phenolic compounds which were largely lacking from the leaf exudate.

A peptide toxin was isolated from a waterbloom of Microcystis aeruginosa from Lake Froylandsvatn in Norway. The isolation procedure included liquid and solid phase extraction and reversed phase high performance liquid chromatography. Amino acid analysis yielded ᴅ-glutamic acid, ᴅ-erythro-β-methylaspartic acid and ᴅ-alanine in equimolar and ʟ-arginine in twofold molar ratios. The presence of dehydroalanine was confirmed by hydrogenation and subsequent amino acid analysis with combined gas liquid chromatography/mass spectrometry. Investigation of the toxin with fast atom bombardment mass spectrometry showed a nominal relative molecular mass of 1023. 3-Amino-9-m ethoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (Adda) was identified by 1 H NMR and 1 H, 1 H COSY spectroscopy. The structure of the toxin was elucidated as 7-desmethyl-microcystin-RR.

Alkaloid extracts from different organs of Adenocarpus hispanicus ssp. hispanicus and Adenocarpus hispanicus ssp. gredensis were analyzed by capillary GC. Twenty-four compounds could be identified by the high sensitive method of GLC-MS: the pyrrolizidine alkaloids decorticasine, N-acetylnorloline and N-butyrylnorloline, the bipiperidyl alkaloid ammodendrine, the phenylethylamine tyramine and 19 quinolizidine alkaloids. In contrast to Adenocarpus complicatus, Adenocarpus foliolosus and Adenocarpus viscosus the alkaloid pattern of Adenocarpus hispanicus is characterized by the occurrence of quinolizidine alkaloids with sparteine predominating in the leaves and numerous dehydroderivatives of sparteine. Remarkable is the total absence of adenocarpine which was described as a main compound of the three former species. Our results strongly support the opinion that the genus Adenocarpus should be divided into two phytochemical groups.

Cell cultures derived from white lupin (Lupinus albus) radicles consist of a mixture of friable cell aggregates and finely divided cells. Both types of tissue accumulate two isoflavonoid aglucones, genistein and 2′-hydroxygenistein, their 7-O-glucosides, and their respective 6-, 8-, and 3′-monoprenyl-, as well as the 6,3′-diprenyl derivatives. Most of these metabolites are differentially released in the culture medium. The relative amounts of aglucones, glucosides and prenylated derivatives that are produced by the aggregates and cells, as well as those excreted into the medium, are discussed in relation to culture growth.

From glandular trichome of Rosa rugosa, three major sesquiterpene hydrocarbons [acora- 3(4),7(15)-diene, carota-4(5),ll(12)-diene (isodaucene) and carota-l,4-diene], and two minor [daucene and acora-3(4),7(8)-diene] were identified. The presence of these carotadienes, which are possibly precursors of the corresponding C-14-oxygenated carotanoids, suggested that the formation of the carotane skeleton followed by the regio-specific oxygenation at C-14 occur in the biosynthesis of R. rugosa carotanoids.

50 U SOD in 1 ml or 10 mᴍ DDTC do not much change sulfite oxidation due to the free radicals procuding system xanthin-xanthin oxidase. Sulfite oxidation due to the activity of this free radicals producing system proceeds probably as the univalent oxidation of sulfite. The role of SOD in SO, resistance mechanism in plants and the role of DDTCas SO 2 oxidation effect stimulator is discussed.

N-Methylanthraniloyl-CoA was synthesized via N-succinimidyl N-methylanthranilate and subsequent transesterification with CoA-SH . This compound was characterized by LSIMS and NMR data. An enzyme preparation from cell suspension cultures of Ruta graveolens catalyzed the formation of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA with a pH optimum of 7.5.

The distribution (or locarization) of lipoxygenase (LOX) and hydroperoxide lyase (HPO lyase) activities in ripening and ripe tomato fruits was investigated. The highest LOX activity existed between skin and outer flesh of tomato fruits. HPO lyase showed no tissue specificity. LOX specifically formed linoleic acid 9-E, Z-hydroperoxide (9-E, Z-HPO) from linoleic acid (LA), whereas HPO lyase specifically cleaved 13-Z, E-HPO. Although a low level (0.36 ± 0.069 nmol/g fr. wt.) of hexanal was detected in the intact tomato fruit, HPOs were not detected. When a tomato fruit was injured by cutting it into 8 fragments and incubated at 25 °C, hexanal increased to 1.642 nmol/g fr.wt. by 30 min. By homogenizing at pH 6.3, hexanal increased to 21.1 nmol/g fr.wt. during a 30 min incubation. UV irradiation of tomato fruits also increased the formation of hexanal. From these results, LOX and HPO lyase are considered to exist as latent forms and to begin the expression of the activity upon injury.

The aspartate aminotransferase from Haloferax mediterranei, which is in the cell mainly as apoenzyme, requires high concentrations of salt for both activity and stability. The maximum activity is reached with 3.5 ᴍ KCl in the assay. The effect of different cations and anions has been studied using several types of salts. Monovalent cations show a significant difference in effectiveness of promoting the activity with the following order: K + > Rb + > Na + > NH 4 + . Mg ++ and polyvalent cations with organic character cause partial activity with maximum effectiveness at 0.1 ᴍ, an inhibition at higher concentration is observed. Anions, added as potassium salts, promote enzyme activity with the following order: Cl - > NO 3 - > I - > SCN - . Like activity, the enzyme stability depends on salt concentrations. Incubation of the enzyme with a low salt concentration leads to inactivation following pseudofirst order kinetics. The inactivated enzyme is partially reactivated by high concentrations of KC1 following second order kinetics. Taking into account the dimeric structure of this enzyme, high concentrations of salt could stabilize the dimer, which is the active form. The salt effects on halophile aspartate am inotransferase are discussed considering hydrophobic and electrostatic interactions.

The antibiotic cerulenin was shown to be a potent dose-dependent inhibitor of de novo fattyacid biosynthesis in intact isolated chloroplasts of different plants (measured as [ 14 C]acetate incorporation into the total fatty-acid fraction). Various chemical derivatives of cerulenin were synthesized and tested in the chloroplast assay-system of oat, spinach and pea. Modifications of the hydrocarbon chain of cerulenin (e.g. tetrahydro-cerulenin and its short-chain cis-2,3-epoxy-4-oxoheptanamide derivative) decreased the inhibitory activity of cerulenin, whereas variations of the epoxy-oxo-amide structural element led to a complete loss of inhibition potency. The results indicate that the naturally occurring antibiotic cerulenin is the most active specific inhibitor of de novo fatty-acid biosynthesis, but the formation of the hydroxylactam ring seems to be an essential requirement for the inhibitory activity. Those structural analogues of cerulenin, which can no longer form a hydroxylactam ring, do not possess any inhibitory capacity.

The nucleus encoded cyanoplast (“cyanellar”) ferredoxin-NADP + -oxidoreductase (FNR ) of Cyanophora paradoxa, characterized by an N-terminal amino acid sequence, is compared with homologous sequences of other photoautotrophic organisms. The high degree of similarity to the cyanobacterial sequences indicates a cyanobacterial origin. This could be a first direct demonstration of an intertaxonic combination: a gene transfer from an original endocytobiont (cyanobacterium) to the nucleus of its host, one of the most important demands of the Endosymbiosis Theory, an evolutionary mechanism leading to the origin of a new species.

Cell suspension cultures of the liverwort, Marchantia polymorpha L. were found useful to study the influence of peroxidizing herbicides either on the greening process or on the fully green cells. The cells of both physiological stages exhibit a characteristic sensitivity to the herbicides. The sensitivity increased rapidly during the exponential phase of growth, reached a maximum during the late exponential phase, and then decreased in the stationary phase. We investigated the kinetics of accumulation of protoporphyrin IX (PPIX) in Marchantia cells treated with several peroxidizing herbicides at various stages of cell growth, and observed a correlation between accumulation of PPIX and herbicidal damage. The glutathione (GSH) content in the cell was also investigated to examine the role of GSH against herbicide treatment. In the light, GSH levels in the cells treated with AFM rose rapidly reaching a peak after 8 h, and rapidly decreased subsequently. The beginning of PPIX accumulation coincided with the decline of GSH after 8 h of treatment. Obviously, GSH plays a key role in protection against oxidative damage caused by AFM in the early treatment period. In the dark, AFM also induced an accumulation of GSH and PPIX, followed by a decline in GSH and PPIX contents during a 20 h incubation. The decline of PPIX was observed several hours after GSH starts to decrease, remaining at a constant level for the following 40 h, leading to accumulation of an other fluorescent still-unknown pigment.

Photosynthetic activity and molecular composition of thylakoid membranes of sensitive and atrazine/paraquat coresistant biotypes of Conyza canadensis (L.) Cronq was compared before and after 5 × 10 -4 molar paraquat spraying. In contrast to the irreversibly damaged sensitive plants the Rfd values (vitality indices) calculated from parameters of fluorescence induction curves, and the in vivo CO 2 fixation in resistant plants showed - after a transitory inhibition - a recovery. Resistant thylakoids contained higher amount of PS II and LHC II, (i.e. lower relative amount of CP I) which can be the result of an adaptation process. Fatty acid composition of total leaf extract was almost the same in both biotype even after spraying. The amount of the oligomeric form of LHCP II decreased in paraquat treated sensitive thylakoids, which was in correlation with the reduction of Δ 3 -transhexadecanoic acid content. Decrease in oligomeric LHCP II and/or special lipids (PG) in sensitive thylakoids may change the association of LHCP II and photosystem II core as it is evidenced from lowering of the Mg 2+ induced changes of the short wavelength fluorescence intensity and increase in the relative quantum requirement values. The results are discussed in connection with a possible effect of paraquat on PS II.

Photosystem II complexes were prepared from chloroplasts of wild type tobacco Nicotiana tabacum var. John William’s Broadleaf and from two chlorophyll mutants derived from it, namely N. tabacum Su/su and N. tabacum Su/su var. Aurea. The hydrophobic peptides of these complexes were analyzed for bound lipid molecules by means of monospecific lipid antisera. A comparison of the peptide composition of the complexes of the three chloroplast types by means of polyacrylamide gel electrophoresis showed that the peptide composition was qualitatively identical. A major quantitative difference referred to a 66 kDa peptide which appeared to be much stronger in gels of photosystem II peptides originating from the yellowgreen and the yellow tobacco variety. Furthermore, we were able to show that different SDS polyacrylamide gel electrophoresis runs of the same PS II preparation yielded differences in the band strength of this peptide. Comparative densitometric measurements showed that an increase in this 66 kDa peptide was always correlated with a decrease in the D 1 and D 2 peptides. Obviously, the 66 kDa peptide is the heterodimer of D 1 and D 2 . Differences in the peptide composition of photosystem II preparations from the 3 tobacco species refer above all to peptides of the light-harvesting complex with molecular masses of 28 and 26 kDa. After the transfer of the peptides from the polyacrylamide gel to nitrocellulose membranes, they were incubated with monospecific antisera to monogalactolipid, digalactolipid or sulfolipid. These experiments showed that the 66 kDa peptide reacted with antibodies to digalactolipid and with those to sulfolipid. The 66 kDa peptide reacts in the Western blot procedure also with an antiserum to a 66 kDa peptide prepared and characterized earlier and which was shown to inhibit electron transport reactions in the region of the reaction center of photosystem II. The monospecific antiserum to monogalactolipid reacts with the D 1 and D 2 peptide as well as with the chlorophyll-binding polypeptides of the masses 42 and 48 kDa, and also with the 26 and 28 kDa peptides of the light-harvesting complex as well as with the extrinsic peptides exhibiting the molecular masses, 33 ,21 -23 and 18 kDa. Whereas lipase treatment apparently destroys the lipids as antigenic determinants of the peptides on the nitrocellulose membrane, periodate treatment or treatment of the photosystem II preparations with organic solvents do not prevent the reaction of the 66 kDa peptide with the sulfolipid antiserum. These experiments show as the 66 kDa peptide appears to be the heterodimer of D 1 and D 2 , that the galactolipids mono- and digalactosyldiglyceride as well as the sulfolipid are bound, much like prosthetic groups, onto the core peptides.

This report demonstrates the ability of folic acid to activate rat liver porphobilinogen-deaminase (PBG -D). Lineweaver-Burk analysis revealed an increase in V max (38%) without affecting the K m . In the concentration range assayed, secondary replots of 1/Δslope and 1/Δintersect versus 1/[folic acid] yielded straight lines, indicating the binding of a single molecule of activator to the enzyme PBG-D , with a K A = 1.66 mᴍ. Results presented here show that folic acid acts as a non-essential activator (α = 1; β = 1.6). The activating effect of folic acid has been observed employing the 35-70% ammonium sulphate precipitated fraction, desalted by dialysis or gel filtration, whereas no action was detected when other partially purified PBG -D preparations were utilized as the enzyme source, suggesting either the presence of sites saturated for the activator, or the existence of a different structural protein conformation, or both.

The effect of chloroquine in combination with bright light on the ultrastructure of crayfish (Orconectes limosus R.) photoreceptors was investigated in vivo. Chloroquine had several effects upon the crayfish retina. Multivesicular bodies (MVB) that are involved in lysosomal degradation of photosensory membrane were altered in ultrastructure. MVB frequently contained smaller MVB in a state of more advanced membrane degradation. Additionally screening pigment-like granules appeared in MVB. MVB accumulated in and filled the retinular cell cytoplasm just proximal or distal to the basement membrane which indicated inhibition of photosensory membrane degradation. Under chloroquine treatment rhabdom degradation appeared to be inhibited, as rhabdom diameter was less reduced under these conditions. Also chloroquine caused accumulation of screening pigment granules in glial cells within the lamina ganglionaris.

The effect of ATP on the calcium release channel in heavy sarcoplasmic reticulum vesicles modulated by ryanodine has been analyzed by monitoring active calcium uptake and caffeine induced calcium release under near physiological conditions. Native as well as ryanodine reacted vesicles display a complex time course of calcium uptake resulting in nearly complete exhaustion of medium calcium when ATP in combination with an ATP-regenerating system, in contrast to ATP alone, or dinitrophenyl phosphate, were used to support calcium uptake. Applying of dinitrophenyl phosphate as energy yielding substrate, not affecting channel activity, allowed to estimate the fraction of light vesicles devoided of calcium channels contam inating the heavy preparation as the fraction that stores calcium after the preparation has been treated with channel opening, low concentrations of ryanodine (1 μᴍ). Calcium uptake by contaminant light vesicles (25%) cannot account for calcium storage, as well as, abolition of caffeine induced calcium release of ryanodine treated heavy vesicles. Calcium uptake of native and ryanodine treated vesicles is accompanied by the uptake of equivalent amounts of inorganic phosphate arising from ATP hydrolysis indicating that calcium is mainly stored as calcium phosphate. The momentary capability of the preparation to accumulate calcium was measured by activating calcium uptake during the calcium storage period with 0.2 mᴍ 45 CaCl 2 and 4 mᴍ phosphate at short time intervals. A significant increase of the momentary uptake activity with time was observed being more pronounced for ryanodine treated than for native vesicles indicating that under regenerating conditions, ATP can induce closing of the native and even more effectively of the ryanodine modified calcium release channels.

Both calcium and caffeine induced calcium release from actively loaded heavy sarcoplasmic reticulum vesicles were studied to analyze the dependence of both activities on the composition of the release medium with respect to monovalent anions. Calcium is unable to induce net calcium release while caffeine remains effective as releasing agent when the experimental media contain neither chloride nor nitrate ions. Caffeine induced calcium release is not suppressed by chelating residual medium calcium (approximately 0 .5 -1 μᴍ) with 2 mᴍ EGTA added 15 s prior to 10 mᴍ caffeine. Calcium release from vesicles loaded in media containing 0.2 ᴍ gluconate as monovalent anion is induced when the medium is supplemented with chloride or nitrate. The release amplitude increases linearly when K-gluconate is replaced by KCl. At constant ionic strength the release amplitude becomes maximal at a chloride concentration of 0.2 ᴍ. The chloride effect completely disappears when 2 mᴍ EGTA are added simultaneously. When chloride is replaced by nitrate, as releasing agent, maximal release is achieved already by addition of 0.1 ᴍ K-nitrate. The releasing effect of nitrate can only partially be suppressed by EGTA. The different effectiveness of gluconate, chloride and nitrate as calcium release supporting ions corresponds to their activating effect on the binding of ryanodine to the calcium release channel in the vesicular membranes.

Corpora allata (CA) from male Locusta migratoria were tested for their ability to synthesize juvenile hormone (JH) and to respond to stimulating brain/corpora cardiaca (CC) extracts under in vitro conditions. We found that a preincubation of the CA of both sexes at 4 °C for 24 h lowers their basal rate of synthesis and retains their responsiveness to allatotropic factors. Male CA can be stimulated by brain/CC extracts as well as female CA. JH biosynthesis stimulating factors are also present in male brain/CC extracts. Thus such extracts from male locusts can be used for the isolation of locust allatotropin. Furthermore male locust CA are appropriate for bioassaying such allatotropic factors.

The distal pigment cells of Orconectes limosus contain two layers of large electron lucent vacuoles that are separated by layers of small right-angled platelets adjacent to the crystalline cones. The crystalline cones of the dioptric apparatus of this species have evaginations into the distal pigment cell cytoplasm. In photoreceptors of Orconectes limosus and Procambarus clarkii a dark pigment accumulation site was detected just distal to the basement membrane at the edges of each retina. These pigment accumulations occurred independent of the state of light adaptation. Ultrastructurally the pigment granules at this accumulation site resemble distal screening pigment granules according to their size (up to 1.2 μm in diameter) and fibrous structure. Distal screening pigment granules were also found in tube-like cell processes or extracellularly within and proximal to the retinal basement membrane, indicating pigment transport to and across the basement membrane. Proximal to the basement membrane screening pigment granules were also observed disintegrated to a gravel-like electron dense material in widely branched cells. Evidence was found that an electron dense material, probably resulting from disintegrating screening pigment granules, was incorporated in the integument of the eyestalk. Four hours after injection of gold particles into the eye stalk distal to the retina they were detected inside and proximal to the retinal basement membrane.

The amperometric analysis of photosynthetic oxygen evolution in thylakoid membrane preparations of tobacco, sedimented on a large surface electrode, shows that the oxygen signal is preceded by a lag phase of 1.2 ms. As the used tobacco lamellar systems have an average diameter of 4 - 5 μm this lag phase is interpreted to be due to an average distance of 2.4 μm between oxygen emitting sources and the electrode surface. This interpretation of the lag phase is substantiated by the fact that photosystem I-mediated oxygen uptake in the same preparation measured with the same amperometric technique exhibits the same lag phase although the kinetic uptake parameters are considerably slower than those of oxygen evolution due to the water-splitting reaction. The analysis of the oxygen evolution signal shows that the sedimentation time i.e. the quality of the contact of the source with the electrode is decisive for the measured value for the oxygen release time, which with this method without much effort comes out to be certainly not longer than 1.5 ms. A computer simulation shows that the distance of the oxygen emitting layer from the electrode as well as its thickness and the actual release time are variables which influence practically all parameters (signal shape, amplitude etc.) of such amperometric signals. From this it appears that the determined apparent experimental parameters of oxygen evolution kinetics contain deformations due to the thickness of the sample and the diffusion distance from the electrode. An extrapolation calculated for a sample of infinite thinness separated from the electrode by a minimal distance of 0.001 μm with τ of 5.5 ms yields a lower limit value for the half-rise time of 0.5 ms, a value which has been experimentally determined by EPR oximetry (K. Strzalka, T. Walczak, T. Sarna, and H. M. Swartz, Arch. Biochem. Biophys. 281, 312 (1990)).

The crystal structure of the title compound (C 40 H 56 , M w = 536.8) has been redetermined by X-ray diffraction methods in order to achieve a structure at higher resolution suitable for theoretical calculations. The crystal system is monoclinic, P2 1 /n, a = 7.656(2), b = 9.445(5), c = 23.536(15) Å, β = 93.41(2)°, V = 1698.8(15) Å 3 , Z = 2, D = 1.050 m g/m 3 , 130 K. R = 0.071, R w = 0.076.

Bud exudates of Populus simonii and Populus yunnanensis were similar and contained primarily caffeic acid and its esters together with the flavanones pinocembrin and pinobanksin-3-acetate. The terpenoid patterns of the specimens examined varied, but this variation was not species related. On the basis of bud exudate analysis P. simonii and P. yunnanensis may be considered to represent a single species.

Aqueous solutions of a yellow and highly fluorescent derivative from dimethyl-POPOP, obtained by treating a chloroform solution of this oxazole scintillator with dimethylsulfate, induce a strong blue-green or yellowgreen fluorescence in chromatin DNA under ultraviolet (365 nm) or violet-blue (436 nm) excitation, respectively. It is suggested that this new and selective fluorescence reaction could originate from binding of the oxazolium derivative into the minor groove of DNA.

In the whole embryo, O 6 -alkylguanine-DNA alkyltransferase (AT) activity increased until day 9 of development and declined sharply after day 13. AT activity of the liver was greatest between day 12 and day 20 and decreased fast after hatching. In the brain, AT activities reached a maximum at day 17 and 18 and declined sharply after hatching. At two developmental stages with different AT activities (day 10 and day 17) DNA alkylation in the brains was estimated 6, 12, 24 und 48 h after administration of N-methyl-N-nitrosourea in ovo by viscometric measurement of DNA fragmentation. The high AT activities of the chicken embryo brain at the 17th day of development correlated with minor DNA fragmentation following a repair period of 12-24 h. It is suggested that the high basal level of AT in the chick embryo might have a protective function against the persistence of the genotoxic lesion O 6- methylguanine during development.