Volume 43, Issue 3-4

In trace analysis it is more and more attempted to replace wet-chemical detection procedures by methods which allow a quantitative analysis without any material disintegration, but on exploiting the characteristic physical properties of the components searched for. Based on a procedure for physical and biological valuation of the air quality by means of X-ray microanalysis (WDX/EDX), a procedure which was previously developed by our group, we deepened our investigations by the help of electron-energy-loss-spectroscopy (EELS). The combination of that procedure with EELS proved to be very advantageous as it revealed a high sensitivity as well as a high energy resolution. The main advantages are to be found in the simple arrangement of the sample detector and in the fact, not only being able to examine deposits microscopically, but also to analyze them chemically without disintegrating the material. Thus loss of material and denaturation are largely excluded.

A quartz based cavity for pressure dependent EPR measurements on liquid samples allowing pressures up to 0.6GPa was constructed. First investigations with this setup were done on spin labeled horse hemoglobin derivatives both in ferric and ferrous state of oxidation. The second derivative EPR spectra show changes of the label’s mobility, which are not correlated with spin state changes of the Fe-porphyrin complex, but which point out structural fluctuations inside the globin protein matrices.

A sensitive and specific method for detection and quantification of methyl phosphonic acid, paminophenyl 1,2,2,-trimethyl-propyl diester (MATP) as a model substance for organophosphorus compounds is described. Different procedures for coupling the haptenic group for immunization, purification and immobilization allowed the detection of hapten-specific antibodies. The competitive inhibition enzyme immunoassay (CIEIA), using purified chicken and rabbit IgG-antibodies, was able to detect MATP-concentrations as low as 10 -10 mol/1. Based upon our results, we postulate that the CIEIA represents a good alternative to the customary diagnosis of organophosphate intoxications, measuring blood Cholinesterase activity.

Quantity and composition of epicuticular leaf wax of Tilia tomentosa Moench. and T. × europaea L. were examined and showed similar wax compositions. The waxes of these two Tilia species contained homologous series of n-alkanes, wax esters, aldehydes, acetates, primary alcohols and fatty acids. In addition to these common epicuticular wax constituents, the triterpenol β-amyrin was found free as well as esterified with long chain fatty acids and in very high amounts with acetic acid in both Tilia species.

A new antimicrobial and cytotoxic sesquiterpenoid, lentinellic acid (1), has been isolated from submerged cultures of Lentinellus ursinus and L. omphalodes. The structure of the antibiotic was elucidated by spectroscopic methods and a single crystal X-ray analysis. 1 may be formed biogenetically by condensation of a protoilludane aldehyde 4 with a malonate unit.

From the roots of Pterocarpus marsupium 7-Hydroxy-6, 8-dimethyl flavanone-7-O-α- L -arabinopyranoside and 7,8,4′-trihydroxy-3′, 5′-dimethoxy flavanone-4′-O-β-ᴅ-glucopyranoside have been isolated and their structure elucidated.

Developing primary leaves of Secale cereale L. exhibit a dynamic metabolism of the major flavonoid luteolin 7-O-[β-ᴅ-glucuronosyl(1→2)β-ᴅ-glucuronide]-4′-O-β-ᴅ-glucuronide (R 1 ). Final steps of R, biosynthesis are sequential glucuronidations of luteolin, which are catalyzed by three specific UDP-glucuronate: flavone glucuronosyltransferases. These enzymes reach highest activities at the fourth and fifth day of leaf development, coinciding with maximal R, accumulation. The activities decrease with advancing age of the leaves. In contrast, a R 1 -specific β-glucuronidase, responsible for the hydrolysis of glucuronic acid in position 4′, shows increasing activity up to the 5 th or 6 th day; but this activity, leading to luteolin 7-O-diglucuronide (R 2 ), is not reduced in later developmental stages. In this phase of leaf development, the amount of R, drastically drops, whereas R 2 accumulates only slightly. From in vitro results and from feeding experiments using [ 14 C]cinnamic acid, a precursor of R, biosynthesis, we conclude that the anabolic sequential glucuronidation takes place in young and expanding leaf tissue, whereas deglucuronidation occurs in nearly mature and mature tissue. The three glucuronosyltransferases as well as the β-glucuronidase, and the flavonoids R 1 and R 2 are localized in the mesophyll.

Chloroplastic glutamine synthetase of Sinapis alba, purified to homogeneity by a simple three step procedure, revealed a molecular weight of about 395 kDa. The native enzyme is composed of eight subunits of identical molecular weight (about 50 kDa (each), although isoelectrofocusing yielded six distinct bands in the pH 5.6 region of the gel. Labelling of the enzyme with the glutamate analogue herbicide [ 14 C]phosphinothricin and with [γ- 32 P]ATP indicated that glutamine synthetase has eight reactive centers per molecule. The native enzyme dissociated into two enzymatically active subaggregates of about 195 kDa after Mg 2+ deprivation.

The cytochrome P-450-dependent monooxygenase digitoxin 12β-hydroxylase from cell cultures of Digitalis lanata needs NADPH and molecular oxygen and hydroxylates cardiac glycosides with the aglycon of digitoxigenin to the corresponding derivatives of the C-series. Other electron donors cannot replace NADPH. The apparent K m -values are 26 μᴍ for NADPH, 7.1 μᴍ for β-methyldigitoxin and 10 μᴍ for digitoxin. The reaction is inhibited by NADP + and cytochrome c in a competitive mode. The optimum temperature was at 20 °C. Low concentrations of Mn 2+ , Mg 2+ , and EDTA were slightly stimulatory, but there was no strict dependence on divalent cations. Digitoxin 12β-hydroxylase is very stable at room temperature and the reaction proceeds for more than 20 h. After the addition of 15% glycerol, 70% of the original activity can be retained subsequent to freezing at -18 °C. By means of linear sucrose gradient fractionation of cellular membranes the digitoxin 12β-hydroxylase was found to be located in the endoplasmic reticulum.

Ferredoxin-NADP + -oxidoreductase (FNR, EC 1.18.1.2) has been shown to be activated by light within a few seconds during dark-light transitions and inactivated in the dark. In previous papers this could be pointed out by the correlation of cytochrome f induction kinetics to the rate of NADP-photoreduction and the variable fluorescence. The present study deals with the role of the proton gradient during the activation process. The transition from an inactive to an active form is followed continuously in an in situ system. The steady-state rate of NADP-photoreduction is affected only by ionophores which inhibit a formation of the proton gradient, but not by inhibitors of the electric field. It correlates to the 9-aminoacridine fluorescence quench and the light scattering signals at 535 nm. The perception of the pH-gradient through the enzyme is still a matter of discussion.

A single binding site for phosphate was found on isolated chloroplast coupling factor in the absence of nucleotides. In our experiments the phosphate binding site showed a K d of 170 μᴍ. We did not observe any differences whether the ATPase activity of CF] had been activated or not. If the enzyme was incubated with [γ- 32 P]ATP the amount of 32 P bound per CF 1 depended on the pretreatment of the enzyme: In the presence of ADP no ATP or phosphate was bound to CF,. After activation of ATPase activity one mol of ATP per mol CF, was rapidly bound and hydrolyzed while there was a slowly occurring binding of another phosphate without concomitant nucleotide binding. We conclude that there are two different types of phosphate binding observed in our experiments: 1) Inorganic phosphate can be bound by one catalytic site per mol of CF 1 2) The γ-phosphate of ATP is able to bind to an ATP binding domain of the enzyme if this domain can exchange substrates with the incubation medium. This ATP binding domain appears to differ from the site binding inorganic phosphate, because at least a portion of the coupling factor contains more than one labelled phosphate during our ATPase tests.

The structure of the ATP-synthase, F 0 F 1 , from spinach chloroplasts and beef heart mitochondria has been investigated by electron microscopy with negatively stained specimens. The detergent- solubilized ATP-synthase forms string-like structures in which the F 0 parts are aggregated. In most cases, the F 1 parts are arranged at alternating sides along the string. The F 0 part has an approximate cylindrical shape with heights of 8.3 and 8.9 nm and diameters of 6.2 and 6.4 nm for the chloroplast and mitochondrial enzyme, respectively. The F 1 parts are disk-like structures with a diameter of about 11.5 nm and a height of about 8.5 nm. The F, parts are attached to the strings, composed of F 0 parts, in most cases, with their smallest dimension parallel to the strings. The stalk connecting F 0 and F 1 has a length of 3.7 nm and 4.3 nm and a diameter of 2.7 nm and 4.3 nm for the chloroplast and mitochondrial enzyme, respectively.

When the sub-core complex of photosystem II, D1/D2, is irradiated at 436 or 415 nm (absorption by chlorophyll and pheophytin and β-carotene) or 540 nm (absorption primarily by pheophytin), the low temperature fluorescence spectrum has two maxima, at 685 and 674 nm. This shows the existence of at least two different fluorescent forms of chlorophyll (chlorophyll a and perhaps pheophytin a). When carotene is irradiated at 485 nm (absorption primarily by β-carotene), only fluorescence at 685 nm is observed: this indicates that carotene is transferring energy to only the long-wavelength form of chlorophyll in the D1/D2 sub-core complex. The band of carotene (at 485 nm) does not appear in the fluorescence excitation spectrum, measured at 674 nm. The position of the carotene molecule relative to each of the fluorescent forms of chlorophyll was determined from the excitation spectra of each of the fluorescence bands.

Chemical and enzymatic procedures have been employed for the preparation of various phosphorylated derivatives of the acyclonucleoside 9-(1,3-dihydroxy-2-propoxymethyl)adenine, an analogue of the active antiviral agent 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG). In combination with the previously reported 2′,3′-seco nucleosides and their phosphates and cyclic phosphates (Stolarski et al., Z. Naturforsch. 41c, 758-770, 1986), this made available a broad class of acyclonucleosides and nucleotides, the acyclic moieties of which are capable of mimicing the ribose and 2′-deoxyribose rings. The solution conformations of the foregoing were determined with the aid of 1 H, 13 C and 31 P NMR, and compared with those of DHPG and 9-(hydroxyethoxymethyl)guanine (Acyclovir, ACV). Particular attention was devoted to conformations about C-O bonds in different acyclic fragments, which demonstrated well-defined differences between 2′,3′-seco derivatives on the one hand (conformational “rigidity”) and derivatives with DHP and AC acyclic chains on the other (rotation about the C(1′)-O(4′) bond). The overall results are in good general agreement with reported crystal structures, and are compared with those obtained by quantum mechanical calculations. The conformational features of the various compounds are also discussed in relation to their substrate and/or inhibitor properties in a number of enzyme systems, including adenosine deaminase, phosphodiesterases, nuclease P1, 3′-nucleotidase and herpes virus type 1 thymidine kinase.

With the nuclear exchange assay and the nuclear retention assay it is shown that the androgen insensitivity of Tfm mice is probably due to a defect of the nuclear acceptor sites for the testosterone receptor complex. Furtheron the results obtained point strongly to the possibility that hormone free androgen receptor is localized in the nuclei and in the cytoplasma according to the “equilibrium model”. A practicable method for separation of unbound steroids from nuclei is described.

Sethoxydim is a postemergence herbicide used to control grass weeds. Application at concentrations higher than 0.1 mᴍ to leaf segments of the sensitive grass species Poa pratensis and Festuca ovina and the tolerant species Poa annua and Festuca rubra, caused a reduction of the electrical membrane potential (⊿E). The depolarization was reversible and depended linearly on the herbicide concentration. The passive diffusion component of ⊿E was not affected by sethoxydim indicating that the herbicide did not change passive permeability characteristics of the plasmalemma. Consequently sethoxydim reduced the active component of ⊿E that depended on primary active transport processes across the plasmalemma. Moreover, sethoxydim increased the reduction of ⊿E of grass leaf cells that was associated with the onset of H+-amino acid cotransport. Simultaneously the uptake of alanine into leaf segments was reduced. From these results it had to be concluded that the plasmalemma-bound H + -ATPase was inhibited by sethoxydim in sensitive and tolerant grasses. In vitro ATP hydrolysis of plasmalemma vesicles isolated from grass leaves by polymer phase partitioning, however, was not inhibited by sethoxydim. Apparently another primary active transport mechanism that may contribute to an electrochemical H + - gradient across the plasmalemma, i.e. a plasmalemma-bound redox system, should be the site of inhibition responsible for the membrane effects of sethoxydim observed in vivo.

Leaf slices of sethoxydim-sensitive (Poa pratensis, Festuca ovina) and tolerant (Poa annua, Festuca rubra) grass species were found to be able to reduce extracellular ferricyanide by electron transport across the plasmalemma. This reduction was inhibited by the herbicide sethoxydim, and the sensitive species were much more strongly affected than the tolerant ones. The inhibition was concentration dependent. Sethoxydim affected the electron transport in isolated Poo chloroplasts. Polarographic and photometric measurements of ferricyanide reduction suggested photosystem II to be the site of action. Inhibition occurred only in the sensitive species Poa pratensis and was linearly dependent on concentration. In isolated mitochondria from sensitive Zea mays coleoptiles sethoxydim also acted as an inhibitor of electron transport. Only slight inhibition was measured with mitochondria from tubers of tolerant potato (Solanum tuberosum). It is concluded that sethoxydim generally acts as inhibitor of membrane-bound redox systems. The specific inhibition of the plasmalemma-bound redox system of sethoxydim-sensitive plants could contribute to a selective action of the herbicide.

We have investigated by means of electron spin resonance (ESR) spectroscopy the influence of three inhalation anesthetics, i.e. halothane, chloroform and diethyl ether, on the interfacial and hydrophobic region as well of 38 mol% cholesterol containing DPPC unilamellar vesicles. The study has been carried out in the temperature range 25-45 °C. The variation of the order parameter, S, vs temperature of the lipid phase indicates that with this content of cholesterol the characteristic gel → liquid crystalline main phase transition of DPPC, normally occurring at T t ~ 41 °C, disappears. When halothane and chloroform are added to the vesicles suspension up to [DPPC]/[anesthetic] molar ratio of 1:1 the main phase transition, as detected with the stearic acid spin label I(12,3), reappears again and it results down shifted at T t ~ 35 and 39 °C, respectively. In presence of diethyl ether, instead, the main phase transition is not observable also at the highest concentration of anesthetic used. Moreover, halothane and chloroform affect similarly the hydrophobic core of cholesterol-!- DPPC vesicles which, in turn, results to be different from the action exerted by diethyl ether in the same region. The ESR findings are discussed in terms of competitive effects shown by cholesterol and inhalation anesthetics. Moreover, the interfacial region of CHOL + DPPC vesicles results to be the target of anesthetics.

The effects of short-time fumigation (0-60 min) of intact spinach leaves with SO 2 (2 ppm) on the photosynthetic apparatus were investigated. A rather high SO 2 concentration was applied to monitor immediate effects on the fluorescence behaviour with the influence of repair processes or secondary types of damage being minimized. Three different types of in vivo chlorophyll fluorescence measurements were used: Rapid induction kinetics (Kautsky effect), slow induction kinetics with repetitive application of saturation pulses (saturation pulse method), and decay kinetics following a single turnover saturating flash. The slow induction kinetics with repetitive application of saturation pulses reacts in the most sensitive way indicating a primary damage at the level of the enzymatic reactions of the Calvin cycle. It is suggested that stromal acidification upon SO 2 uptake interferes with light activation of Calvin cycle enzymes. With longer fumigation times also damage at the level of photosystem II becomes apparent: A decrease in variable fluorescence yield reflects a lowering of photosystem II quantum yield, and the slowing down of fluorescence relaxation kinetics reveals an effect on the secondary electron transport from Q ᴀ to Q в . The detrimental effects of SO 2 depend to a great extent on the application of light during fumigation. Besides a light requirement for SO 2 uptake by stomata opening also the possibility of photoinhibitory damage is discussed. The susceptibility of leaves to photoinhibition may increase with a lowering of Calvin cycle activity by SO 2 .

In living antennae of the male silkworm moth Antheraea polyphemus the pheromone compounds [6,7- 3 H]-6,11-hexadecadienyl acetate and [12,13- 3 H]-10,12-hexadecadienol are enzymatically converted to their corresponding fatty alcohols, aldehydes, acids and long-chained fatty acid esters. In antennae of freshly hatched moths the 3 H-labeled pheromones are degraded at high rates to volatile polar metabolites. The half-life of the pheromone acetate is about 3 min. In dried antennae the pheromone acetate is merely hydrolyzed to the fatty alcohol.

ESR studies on spin-labeled amorphous RNase A as a function of varying concentrations of sorbed H 2 O and D 2 O will be presented. A relaxation analysis of saturation transfer (ST-)ESR spectra of 14 N( 1 H) nitroxide spin-label molecules essentially fixed at amino acid residue His-105 will be given. A characteristic correlation has been observed between the microdynamic behavior - expressed by the rotational correlation times of the paramagnetic label - and the macroscopic thermodynamic entropy for the sorption process of H 2 O and D 2 O at RNase. This correlation is particularly pronounced at low water concentrations, viz., n H₂O /n protein ≤ 100. A significant difference in this concentration range exists between the two systems “RNase-H 2 O” and “RNase-D 2 O”, which is manifested not only by the thermodynamic data but also by the microdynamic behavior extracted from the corresponding non-linear ESR absorption line shapes.

In Xenopus, the supraorbital lateral line system consists of a periodic pattern of lateral line organs which is formed by the regular fragmentation of a streak-like primordium. The pattern forming mechanism which subdivides the primordium into individual organs is not capable of adjusting to the variable size of the system. Nevertheless, the number of organs per supraorbital system tends to be held constant. This is achieved by regulating the growth of the system in an appropriate manner.

Propolis balsam from four locations in Oxfordshire was analysed. The balsam was qualitatively similar but showed large quantitative differences in composition. These quantitative differences are related to the bud exudate composition of the poplars from which balsam was gathered.

2′,5′-Dihydroxyflavone and its 5′-acetate were isolated from the farinose exudate of Primula japonica and P. pulverulenta. Their structures were elucidated by spectroscopic methods and confirmed by synthesis. Both flavones are novel natural products.

A study on distribution of hydroperoxide lyase activities in cucumber seedlings was performed. Hydroperoxide lyase activities were at elevated level in root, and in the upper and greener sections the lower activities were observed.

Columnidin, the characteristic 3-deoxyanthocyanidin of some Columnea species, possesses the 3′,4′-B-ring hydroxylation pattern of luteolinidin and an additional hydroxyl group at the A-ring, most likely in the 8-position. Studies on substrate specificity of chalcone synthase and flavanone 4-reductase and the demonstration of high flavonoid 3′-hydroxylase activity revealed that the 3′-hydroxyl group of the B-ring of columnidin is introduced at the flavanone stage by hydroxylation of naringenin to eriodictyol. Enzymatic hydroxylation of the A-ring, however, could neither be observed with soluble enzyme preparation nor with microsomal fraction. Most probably this step first occurs at the anthocyanidin level. Besides flavonoid 3′-hydroxylase the microsomal fraction of Columnea flower extracts contains flavone synthase II activity catalysing desaturation of flavanones to flavones with NADPH as co-factor. The presence of some apigenin, appreciable amounts of luteolin and of the 3′,4′-hydroxylated flavan-4-ol luteoforol in the flowers confirm the enzymatic data.

(Z)-9-Dodecen-7-yn-1-yl acetate (y7Z9-12Ac) (3) is suggested as a pheromone mimic for Lobesia botrana. It was synthesized in two different ways and its activity was demonstrated electrophysiologically and in behavioural laboratory and field experiments.