Volume 42, Issue 7-8

A psbA gene fragment has been cloned into an expression vector in frame with the 3′end of the beta-galactosidase gene. Expression of this construct in E. coli results in an overproduction of a hybrid protein consisting of part of the herbicide binding protein and beta-galactosidase. An antiserum raised against this fusion protein specifically detects a 34 kDa polypeptide within the complex mixture of spinach thylakoid membrane proteins.

Department of Genetics, The Hebrew University of Jerusalem. Jerusalem. A variety of mutants which are resistant to triazine - and urea - classes of herbicides have been isolated in the cyanobacterium Synechococcus R2. All the mutants that have been analyzed, show some cross-resistance to different herbicides suggesting that these herbicides share a common binding site in photosystem II. Three psbA genes have been identified in Synechococcus R2. The psbA-copy I gene was cloned from three mutants and used in DNA-mediated genetic transformation. It was found that in all three mutants this gene could transfer the mutation for herbicide resistance indicating that this gene codes for the herbicide resistant protein.

Site-directed mutations were created in the cyanobacterium Synechocystis 6803 to alter specific histidine residues of the photosystem II (PS II) D2 protein. In one mutant (tyr-197). the his-197 residue was replaced by tyrosine, in another mutant (asn-214), his-214 was changed into asparagine. The tyr-197 mutant did not show any low-temperature fluorescence attributable to PS II. but contained a PS II chlorophyll-protein, CP-47, in significant quantities. Another PS II chlorophyll-protein, CP-43, was absent, as was PS II-related herbicide binding. The asn-214 mutant showed a blue-shifted low-temperature fluorescence maximum around 682 nm. but did not have a significant amount of membrane-incorporated CP-43 or CP-47. Herbicide binding was also absent in this mutant. These data indicate a very important role of the his-197 and his-214 residues in the D 2 protein, and are interpreted to support the hypothesis that the D2 protein and the M subunit from the photosynthetic reaction center of purple bacteria have analogous functions. According to this hypothesis, his-197 is involved in binding of P680. and his-214 forms ligands with Q ᴀ and Fe 2+ . In absence of a functional D2 protein, the PS II core complex appears to be destabilized as evidenced by loss of chlorophyll-proteins in the mutants.

Oligonucleotide-mediated mutagenesis has been used to change isoleucine 229 of the reaction center L subunit of Rhodopseudomonas capsulata to seventeen other amino acids. A system of deletion strains and complementing plasmids has been used to assay the effects of changing this residue, which forms part of the secondary quinone O в binding site. The reaction center is assembled in all seventeen mutants. Ten of the seventeen mutations (Gly, His, Tyr, Trp, Asn, Asp. Gin, Glu, Arg, and Lys) result in the loss of photosynthetic growth. The remaining seven mutations yield varying levels of photosynthetic growth (Ile, Val, Ala, Leu, Met>Thr, Cys, Ser). Six of these mutations lead to varying atrazine resistance (Met, Leu > Ser, Ala, Thr, Cys>Val, Ile). None of the mutations lead to atrazine sensitivity. These results suggest that hydrophobic residues of moderate size function best at this position. This set of mutations at isoleucine L229 include the first examples of mutants with null quinone function and the first examples of crystallography-based genetic engineering in the design of herbicide resistant reaction centers.

The objectives of this study were to determine if there were variations in triazine-resistant and -susceptible Brassica napus leaf disc chlorophyll fluorescence (LCF) intensity in terms of: age of leaf on the plant and time of day. In growth room and field experiments triazine-susceptible B. napus cv. “Tower”, and triazine-resistant B. napus cv. “OAC Triton” were used. Chlorophyll fluorescence intensity measurements were made 30 min after disc removal. In both environments, two periods of reduced photosynthetic efficiency occurred in the diurnal. The times that these periods occurred during the diurnal differed between biotypes. A phase shift in LCF maxima between resistant and susceptible biotypes resulted in two periods, early and late in the light period of the diurnal, of increased LCF in resistant tissue. This differential pattern in LCF is support for the hypothesis that triazine resistance chloroplast alterations could imply an alteration in the temporal organization of chloroplast physiological function.

Triazine resistance in a mutant biotype of Phalaris paradoxa is accompanied by changes in the chlorophyll fluorescence induction curve, and by reduced quantum yield for electron transport, indicating altered photosystem II activity. However, light-saturated rates of electron transport in isolated chloroplasts, rates of CO 2 uptake in leaves and dry weight production of the triazine resistant biotype, are equal or superior to those of the wild type. A single mutation in the psbA gene, leading to a serine to glycine shift at position 264 of the thyiakoid membrane 32 kDa Q в - protein. was found in the herbicide resistant mutant. The results indicate that triazine resistance is not necessarily linked to inferior photosynthetic and growth performance.

A comparison of the photosynthetic activities, chloroplast ultrastructures and sensitivities to some urea herbicidcs of atrazine-resistant (R) and suspectible (S) biotypes of Conyza canadensis Cronq (L.) (horseweed) in the rosette stage showed that: 1) The light-intensitv dependences of the rates of CO : reduction in intact leaves of the R and S plants are very similar. 2) The rate of the Hill reaction of the R chloroplasts at 25 °C is 15 - 20% lower at all light intensities than that of the S chloroplasts. 3) The rates of the Hill reactions of the R and S chloroplasts exhibit different temperature-dependences. The activation energy for the S chloroplasts in the temperature range 10-40 °C was found to be 15.3 kJ mol -1 . For the R chloroplasts. the discontinuity in the Arrhenius plot was found at around 20 °C; the activation energy was 20.1 kJ mol -1 at below 20 °C. and decreased to 8.2 kJ mol -1 at higher temperatures. 4) The R plants have a better adaptive capacity than the S plants at low temperatures. 5) The chloroplast ultrastructure in the R plants was not reminiscent of that of plants grown under low light intensity. 6) The R biotvpe exhibited cross-resistance to chlorbromuron and metobromuron. and tolerance to linuron. The R and S biotypes, however, were equally sensitive to diuron. The results permitted conclusions on the properties of the triazine-resistance and urea cross-resistance.

The heterogeneity of photosystem II with respect to a and β centers was investigated in triazine-resistant and susceptible biotypes of Chenopodium album. In both biotypes the light harvesting antenna sizes of photosystem II a centers was larger than those of β centers. In the resistant biotype the antenna size of the a centers was smaller than those in the susceptible one. There was not much difference in the antenna sizes of the β centers. The proportion of β centers was larger in the resistant biotype compared with the sensitive one.

Triazine-resistant (R) biotypes of several dicot weed biotypes have been shown to differ from triazine-susceptible (S) biotypes both structurally and biochemically. To this point, no R monocot has been examined, however. In this report, we examine the ultrastructure and physiology of three biotvpes of Poa annua L.: a S biotype, a R biotype, and a “mixed” (M) biotype that displays a level of resistance just slightly less than the R biotype, as well as a unique fluorescence in the so-called M region of the fluorescence induction curve. Like the dicots investigated previously, the R and M biotypes have larger grana stacks, and more light-harvesting chlorophyll alb protein than the S biotype. These are similar to the differences described between “shade-type” and “sun-type” chloroplasts although anatomical parameters, that normally vary between sun and shade type plants, do not appear to be different in the three biotypes. These data indicate that the structural and biochemical modifications of the chloroplast reported previously in R biotypes of dicots are also found in R biotypes of monocots and further indicate that these are natural consequences of the slower electron transport found in R biotypes.

In vitro studies of the influence of FMC 57020 on terpenoid biosynthesis have shown that this herbicide acts at an early stage. It affects the conversion of isopentenyl pyrophosphate to geranylgeranyl pyrophosphate catalyzed by isopentenyl pyrophosphate isomerase and prenyl transferase. An inhibition of the carotenogenic enzymes phytoene desaturase, ζ-carotene desaturase, and lycopene cyclase could be excluded. Comparison of I 50 values for in vivo chlorophyll, carotenoid, ergosterol and gibberellin biosynthesis as well as in vitro formation of phytoene. phytol and kaurene in various autotrophic and heterotrophic organisms have shown that terpenoid biosynthesis in the chloroplast is much stronger affected than extraplastidic terpenoid formation.

The pyridazinone BAS 13-338 (SAN 9785|) inhibits the desaturation sequence leading to polyunsaturated fatty acids, mainly of glycolipids. Parallel to the inhibition of fatty acid desaturation in the presence of the pyridazinone, changes in energy-distribution parameters have been observed. These data indicate that the amount of polyunsaturated fatty acids in glycolipids is strongly correlated with excitation, trapping, grouping and dissipation, but not with spillover. Functional changes in energy distribution induced by BAS 13-338 are interpreted as a consequence of structural changes in the lipid matrix, which may imply a structure-function relationship between pigment protein complexes and the surrounding lipid environment in thylakoids.

The para-nitro substituted diphenyl ether herbicides which cause rapid plant pigment photo-bleaching can be divided into two categories: 1. those that have a photosynthetic requirement for activity (e.g. oxyfluorfen) and 2. those that have no apparent metabolic requirement for activity (e.g. acifluorfen). A model is presented for the latter category, in which the diphenyl ether herbicide interacts with carotenoids and/or chlorophyllide in plastid membranes to form a complex which photochemically generates singlet oxygen or lipid-peroxidizing radicals.

Peroxidative activity of oxyfluorfen and binding of this nitrodiphenyl ether to cell fractions was investigated with the susceptible alga Scenedesmus acutus and the resistant alga Bumilleriopsis filiformis. Although a 10-fold higher concentration of oxyfluorfen was applied to Bumilleriopsis, the lag phase for initiation of peroxidative evolution of short-chain hydrocarbons from fatty acids was much longer than found with Scenedesmus. Oxyfluorfen was predominantly recovered after homogenization from the pellet which was separated into a lipid and a chloroform/methanol insoluble fraction. Parts of the oxyfluorfen which is present in the insoluble pellet fraction during the lag phase before the onset of peroxidation can be found in the lipid fraction when measurable peroxidative activities have started. This was observed with Scenedesmus as well as with Bumil-leriopsis. During peroxidation initiated by oxyfluorfen acyl lipids are degradated as monitored by the disappearance of the plastidic sulfolipid. Analysis of bound fatty acids showed that they are targets for peroxidative reactions in acyl lipids. Destruction of polyunsaturated fatty acids was higher than for saturated ones.

Six pyridyl derivatives [benzylviologen, 2-anilinopyridine, 1,2-bis(4-pyridyl)ethane, 1,2-bis(4- pyridyl)ethylene, 2-benzoylpyridine, and 2-benzylaminopyridine] and five heme-iron derivatives [hemoglobin, hemin, hematin, ferritin, and ferrocene] were screened for their potential to coun- teract paraquat (1,1′-dimethyl-4.4′-bipyridinium ion) toxicity on pea (Pisum sativum L.) isolated chloroplasts. The H 2 O -> methylviologen(MV)/O 2 and H 2 O → ferredoxin(Fd)/NADP + were two Hill reactions assayed with these compounds. Antagonists of paraquat toxicity should inhibit the first Hill reaction but not the latter. All pyridyl derivatives examined did not inhibit the reaction H 2 O → MV/O 2 . Ferritin and ferrocene were also ineffective as inhibitors of this reaction. Hemoglobin inhibited the reaction H 2 O → MV/O 2 without inhibiting the reaction H 2 O → Fd/NADP + , providing protection to pea chloroplasts against paraquat. Hemin and hematin inhibited both Hill reactions examined. They also inhibited H 2 O → diaminodurene(DAD) ox and durohydro-quinone → MV/O 2 Hill reactions but not the dichlorophenol indophenol red → MV/O 2 and DAD red → MV/O 2 Hill reactions. These results suggest that hemin and hematin are inhibiting the photosynthetic electron transport in the plastoquinone-pool region.

A leaf-disc chamber has been used to measure changes in photosynthesis by soybean leaf discs which have been supplied with photosystem I (PS I) electron acceptors via the transpiration stream. The uncharged heteropentalenes induce an inhibitory effect on CO 2 fixation which is dependent on the n-octanol-water partition coefficient of these molecules. Paraquat, which carries a di-positive charge, was ineffective under the same conditions and at a concentration of 10 -4 m. Cationic species such as K + , Ca + and Mg 2 + greatly enhanced the effect of paraquat on CO 2 fixation while the presence of these ions did not affect inhibition by the heteropentalenes. Such differences between uncharged and charged PS 1 electron acceptors have been explained in terms of electrostatic interactions encountered by paraquat on its way to the site of action within the chloroplast. via the transpiration stream.

Pyruvate-phosphate dikinase (PPDK) was targeted for inhibitor design because of its central role in C 4 -type photosynthetic carbon metabolism and its relative absence in C 3 -type plants and organisms of the animal kingdom. Compounds were tested for their activities as reversible and irreversible inhibitors of PPDK isolated from maize. The relative activity of the test compounds against phosphoenolpvruvate carboxylase (PEPCase), isolated from wheat, was used as a measure of the specificity of the inhibitors for the targeted enzyme. PPDK. An ideal compound for successful herbicide design should be an inhibitor which is specific toward the targeted enzyme and either effectively inhibits enzyme activity at very low concentrations or inactivates the enzyme through an irreversible mechanism. Using results obtained with commercially purchased compounds. the discussion will emphasize the difficulty of obtaining specific inhibitors toward PPDK. Another report from this laboratory (A. L. Lawyer. S. R. Kelley, and J. I. Allen, in: Proc. Seventh Internat. Congr. Photosynth. (J. Biggins, ed.), in press) emphasizes other results obtained from the targeting of PPDK for herbicide activity.

Spinach RuBP carboxylase-oxygenase (Rubisco) purified from the commercially available enzyme, underwent kinetic changes when micromolar concentrations of O-p-nitrophenylhy-droxvlamine (NPHA) were applied on fully activated enzyme. Under steady-state conditions, a decrease of K m CO 2 was observed without changes in V max . Double reciprocal plots showed a competitive stimulation of carboxylase function. The substrate specificity factor k = V c K o /V o K c was increased from approximatively 15-20% upon treatment with NPHA, using both parallel and simultaneous determinations of RuBP-carboxylase and oxygenase activities. Such a limited change in partition between Rubisco functions actually resulted into an increase in CO 2 fixation of intact soybeans leaves treated with NPHA. arising from a substantial alleviation of O 2 -inhibition of photosynthesis. From spectrophotometric and sulfhydryl group titration data, evidence was accumulated for a conformational change of the native enzyme, induced by NPHA. bringing about an increased ratio of carboxylase to oxygenase activities. Direct interaction of NPHA with a cysteine residue of Rubisco near the catalytic site, appears as a candidate for such chemically-induced conformational modification.

The photochemistry of C-phycocyanin has been studied after denaturation in the dark. It shows an irreversible reaction which has characteristics of a Z, Z, E- to Z, Z, Z-isomerization of dihydrobilins. Its amplitude depends on the reaction conditions, with a maximum corresponding to 15% conversion of one of the three PC chromophores. This chromophore is suggested to be β-153, for which recent X-ray data T. Schirmer, W. Bode, and R. Huber, J. Mol. Biol., submitted, show ring D being highly twisted out of the plane of the other rings. During unfolding, there is thus a probability of falling into the photochemically labile Z, Z, E-configuration.

Prolonged UV irradiation of ceroptene yielded substantial quantities of a compound identical with the previously described isoceroptene, a trace constituent isolated from the farinose exudate of Pityrogramma triangularis. The improved MS, 1 H NMR and 13 C NMR analyses, together with X-ray diffraction, made possible by the availability of synthetic isoceroptene has led to a revision of the originally proposed structure. Isoceroptene is shown to be a novel, centrosymmetrical dimer of ceroptene, and accordingly the more appropriate name, α-diceroptene, is suggested to replace that of isoceroptene. An improved method for the synthesis of ceroptene is also described.

The absolute configuration of two hydroxybiflavanonols from Garcinia cola nuts have been determined by CD and 500 MHz 1 H NMR spectroscopy. Additionally the occurrence of atrop-isomers at room temperature as the consequence of rotational hindrance in the molecules could be demonstrated.

“Epicuticular waxes” from pine pollen and from pollen wings have been investigated. Various hydrocarbons, aldehydes, wax esters, free fatty acids and primary alcohols in the form of homologous series were identified. A noteworthy observation was the presence of a large number of unsaturated fatty acids. Although there were quantitative differences between the waxes from whole pollen and pollen wings, the distribution patterns of chain lengths of the individual compounds were very similar. It is demonstrated unequivocally, through the use of pollen wing material, that the waxes are components of the exine. It is postulated that the pollen waxes provide an additional effective mechanism for protection against the effects of the physical and chemical environment.

From the moss Bryum capillare (Bryaceae) two new biflavonoids-bryoflavone and hetero ­ bryoflavone - have been isolated. They are the first examples of a new class of biflavonoids formed by oxidative coupling of a flavone and an isoflavone moiety. The structures of both compounds are proved spectroscopically.

[ 14 C]Cadaverine was applied to leaves of Lupinus polyphyllus, L. albas, L. angustifolius, L. perennis, L. mutabilis, L. pubescens, and L. hartwegii and it was preferentially incorporated into lupanine. In Lupinus arboreus sparteine was the main labelled alkaloid, in L. hispanicus it was lupinine. A pulse chase experiment with L. angustifolius and L. arboreus showed that the incorporation of cadaverine into lupanine and sparteine was transient with a maximum between 8 and 20 h. Only leaflets and chlorophyllous petioles showed active alkaloid biosynthesis, whereas no incorporation of cadaverine into lupanine was observed in roots. Using in vitro organ cultures of Lupinus polyphyllus, L. succulentus, L. subcarnosus, Cytisus scoparius and Laburnum anagyroides the inactivity of roots was confirmed. Therefore, the green aerial parts are the major site of alkaloid biosynthesis in lupins and in other legumes.

The vanadium content of different mushrooms of the genus Amanita was measured by atomic absorption spectroscopy. Amanita regalis and Amanita velatipes showed high levels of vanadium comparable with the level present in the fly agaric Amanita muscaria. Electron paramagnetic resonance and high pressure liquid chromatography showed that in these three species amavadin, a vanadium(IV)complex with two N-hydroxy-2,2′-imino dipropionate ligands, was present. In the other species examined, only low levels of vanadium were found.

Spruce Decline, Ascorbic Acid, Glutathione Green and bleached needles or needle segments were analyzed for their ascorbic acid and glutathione contents. All bleached needles or segments exhibited higher amounts of both sub­ stances as com pared to green controls. The physiological significance of the increase in ascorbic acid and in glutathione is discussed in content with their roles as antioxidants.

Naturally-occurring hydroxybutinylbithiophene derivatives were acylated by enzyme preparations of Tagetes patula seedlings in the presence of distinct acyl-Coenzyme A esters. The O-acyltransferase activity could only be detected after almost complete separation of the enzyme from counter-currently acting esterases which were present in the same extracts. This was achieved by affinity chromatography on Cibachron Blue A. During this procedure, the O-acyl-transferase was split, yielding two active fractions. Both had a M r of 37,000 (±5,000), equal isoelectric properties, a pH optimum of pH 7.0, and were considerably inhibited in the presence of free Coenzyme A. Small differences existed in their affinities for their thiophenic substrates (3,4-dihydroxybutinylbithiophene and 4-hydroxybutinylbithiophene, respectively), as well as for various acyl-CoA esters as cosubstrates. With the preferred cosubstrate, acetyl-CoA, acylation took place at the 4-position of the butinyl side chain of the molecules, forming the naturally- occurring 4-acetoxybutinylbithiophene and 3-OH,4-OAc-butinylbithiophene, respectively. From the other acyl-CoA esters employed, only propionyl-CoA was likewise converted, forming the corresponding O-propionyl esters. The reactions observed are suggested to be catalyzed by two acetyl-CoA: 4-hydroxybutinylbithiophene O-acetyltransferase isoenzymes which exhibit different affinities for particular substrates and cosubstrates. The activities of both the isoenzymes changed drastically if plant material from different developmental stages was used as enzyme source. Therefore, it may be suggested that these isoenzymes play an important regulatory role in the metabolism of naturally-occurring hydroxy- and acetoxybutinylbithiophenes and their derivatives.

Growth of several cyanobacteria was examined on ethane sulfonate and taurine as only sulfur source. Comparing two strains with differential utilization of sulfonic acids (Anabaena variabilis and Synechococcus 6301) demonstrated that actual growth was coupled to the presence of an active sulfonate transport system due to species specific properties and nutritional conditions. Sulfonate uptake in Anabaena variabilis was characterized by a pH optimum of 6.5, a structural specificity for sulfonates, missing Na + dependence, and phosphate stimulation. Radiolabeled ethane sulfonate and taurine was metabolized to products of normal sulfur metabolism. Also considerable amounts of 35 S-labeled volatiles (mercaptanes and sulfide) could be detected, suggesting a degradation mechanism via reduction to mercaptanes and cleavage of the C-S bond.

Crude protein extracts from the chickpea (Cicer arietinum L.) pathogenic fungus Ascochyta rabiei catalyze the NADPH dependent conversion of the pterocarpan phytoalexins medicarpin and maackiain to 1) 2′-hydroxyisoflavans, 2) 1 a-hydroxypterocarp-1,4-diene-3-one, and 3) 10,2′-dihydroxyisoflav-8-ene-7-one derivatives. A pterocarpan : NADPH oxidoreductase which cleaves the benzylphenyl ether bond of the phytoalexins was purified some 100-fold and partially characterized with regard to its kinetic properties. The oxidoreductase was shown to be specific for NADPH and 3-hydroxypterocarpans. The enzyme appears to be constitutively expressed by A. rabiei.

Cyanobacterium . Anabaena variabilis, Nitrogenase Regulation, Ammonia, Methylamine Using the heterocystous cyanobacterium A nabaena variabilis (ATCC 29413) in an alkaline environment its nitrogenase activity is rapidly inhibited by ammonia and methylamine. Nitro­genase inhibition by ammonia is probably caused by a mechanism comparable to the switch-off effect, which has been described for several species of the Rhodospirillaceae, whereas methyl-amine-induced inhibition is caused by an uncoupling effect only. Evidence for these different effects is obtained by comparing nitrogenase activity in cell-free extracts of filaments pretreated by ammonia or methylamine. In addition, ammonia-dependent nitrogenase inhibition is shown to be dependent on protein synthesis and on light intensity.

Glucose dehydrogenase from Bacillus megaterium exists as a stable, active tetramer at pH 6.5. By shifting the pH to 9. the enzyme is. completely and reversibly, dissociated into four inactive protomers. Kinetics and mechanism of this pH-induced dissociation have been studied, at various enzyme concentrations, by ultraviolet absorption, circular dichroism, normal and stopped-flow fluorescence as well as by light scattering and activity measurements. Dissociation of the fully active tetramer proceeds via three distinct kinetic steps: (1) fast conformational rearrangement of the tetramer, without any loss of activity (t 1/2 0.0075 sec); (2) slow isomerization to a tetramer with lower specific activity (t 1/2 27 sec): (3) subsequent dissociation of this rearranged tetramer into inactive monomers (t 1/2 114 sec) with still intact native secondary structure. All three processes follow first-order kinetics. Both rate and extent of the dissociation are reduced, with a concomitant shift to higher reaction orders, by increasing the NaCl concentration in the buffer. This suggests the establishment of a dissociation/association equilibrium, due to the concentration- dependent stabilization of the tetrameric enzyme state by NaCl.

Crystal structure data on hydroxyapatite, octocalcium phosphate and brushite have been used in order to predict their crystal morphology on the basis of the Hartman-Perdok theory. The predicted forms are pencil-like for hydroxyapatite, board-like for octocalcium phosphate and flattened needle-like for brushite. Although the biominerals in bone, dentine and tooth enamel have an apatite structure, their form is nbt pencil-like. This may partially be due to the fact that precursor phases are nucleated first in these tissues during mineralization and that these precursor phases are transformed later by topotactical reactions into compounds with apatite structure or that they serve as nuclei for ongrowth of apatite. The form of the mineral particles in mature bone and dentin is board-like which indicates that octocalcium phosphate might be their precursor phase. However, the form of the crystals in mature enamel is flattened needle-like which indicates that brushite is their precursor phase. It is argued that a possible difference in the nature of the precursor phase may be due primarily to differences in the cellular activities of the odontoblasts and osteoblasts as compared to those of the ameloblasts, and secondarily to matrix effects. In both cases, however, the main effect of the matrix seems to be that it acts as a mechanical barrier leading to a limited form and size and, of course, a certain orientation of the crystals, rather than as an agent for heterogeneous nucleation. The validity of the present considerations depends as yet on the assumption that the ions and molecules occurring in body fluids do not dominate the habit of calcium phosphate crystals.

The spin labeled nitrosoureas 7a-e and 12 were synthesized and evaluated in vivo for their anticancer activities against the murine lymphocytic leukemia P388. Compounds 7a-c, 7e and 12 possessed activities ranging from 31 to 542 percent increase in life span (%ILS), whereas compound 7d was marginal (%ILS = 21). All CD 2 F 1 male mice treated with the most active com- pounds (7a and 12) at 35 mg/kg for 9 days were alive after 30 days, whereas all mice treated with the clinical drug CCNU (1c) succumbed. Compounds 7a-e and 12 were further evaluated for their antineoplastic activity against lymphoid leukemia L1210. Compounds 7a and 12 exhibited, on day 60, a %ILS of 713 and 620, respectively. The lipophilicities of compounds 7a-e and 12 were determined using the EPR and UV methods. Compounds 7a and 12 which differ from CCNU and MeCCNU by the replacement of the cyclohexyl and methylcyclohexyl groups with six and five membered nitroxyl radical moieties were more hydrophilic than the clinical drugs.

The primary interaction of cultured Tetrahymena cells with a hormone (insulin in the present case) gives rise to hormonal imprinting, which accounts for a considerable increase in the later hormone binding capacity of the cells. Mixed culturing of imprinted and not imprinted (virgin) cells results in transmission to the latter of the information mediated by imprinting and thereby in a considerable increase in hormone binding capacity over that of pure hormone-preexposed cell cultures. The material substrate of intercellular information transmission is a cellular secretion which appears in the nutrient medium, and is increasingly released in presence of not imprinted (virgin) cells in the mixed culture.

The minimal topological difference (MTD) method is used to describe quantitative structure-activity relationships (OSAR) for the progesterone-receptor binding affinity including 59 proge-stational steroids. Multiple correlation coefficients of r = 0.962 and r = 0.955 are obtained by use of the MTD variable and a measure of hydrophobicity for the series of progesterone and ethisterone derivatives, respectively. Hydrophobic effects are found to strongly influence receptor binding. In accordance with the hydrogen bonding concept, the optimized MTD receptor maps indicate cavity vertices in the regions of oxygen functions at C3 and in the 17β position. Receptor wall vertices are attributed in the areas of 4, 10β, and 13β substituents of 4-en-3-one steroids while 17α side chains additionally contain receptor cavity vertices. A comparison of corresponding receptor maps suggests in accord with X-ray crystal structure data that progesterone and ethisterone derivatives are bound in somewhat different orientations relative to the receptor surface.

Some of the presumable heat shock proteins will be produced in Bacillus subtilis in response to different environmental conditions, e.g. heat shock, amino acid limitation or oxygen limitation. During amino acid limitation or during oxygen limitation the relA + strain is able of synthesizing this set of proteins but the relA strain is not. We suggest that the accelerated rate of the synthesis of some heat shock proteins depends on the induction of the stringent response because the (p)ppGpp production does not occur in the relA strain during amino acid or oxygen limitation. On the other hand the relA strain can produce heat shock proteins under heat stress. Therefore different mechanisms must be responsible for the expression of this set of genes during heat and other stress stimuli. It can be supposed that in B. subtilis the (p)ppGpp-dependent stringent control is a central defense reaction against different adverse environmental conditions and furthermore, that the synthesis of “stress” proteins as an essential component of the stringent response is part of a general adaptation mechanism under non-growing conditions.

Volatile egg secretions of 15 species belonging to 5 families of the brown algal order Laminariales have been analyzed by high resolution glass capillary gas chromatography. 16 components have been identified and partially determined quantitatively. The composition of the pheromone bouquets and pheromone specificity for induction of spermatozoid-release and -chemotaxis are discussed in respect to ecophysiological functions and significance as chemo- taxonomic markers.

The Dufour glands of Leptothorax acervorum and L. nylanderi both contain species-specific mixtures of hydrocarbons, dominated by C 17 hydrocarbons in L. acervorum and C 15 hydrocarbons in L. nylanderi. Both species contain the sesquiterpenoid tetramorene-2. but neither contain (E)-β-farnesene, which has earlier been found in the glands of Harpagoxenus sublaevis which raids Leptothorax nests and enslaves its workers. The contents of the glands of workers and queens of L. acervorum were very similar.

Biosynthesis of the major sex pheromone components of the obliquebanded leafroller, (Z)- and (E)-11-tetradecenyl acetates, is shown to proceed by Δ11 desaturation of myristate as in the related redbanded leafroller. A comparison between the amounts of deuterium label incorporated into the pheromone components from labelled myristic, palmitic and stearic acids gave a higher level of incorporation for the shorter chain acids, suggesting that Δ11 desaturation is a faster process than 2 carbon chain-shortening by β-oxidation.

O-(p-nitrophenyl)-hydroxylamine affects the size of the Warburg effect measured in leaves of the tobacco aurea mutant Su/su. If leaves are treated with a 10 -5 м aqueous solution of the hydroxylamine derivative the Warburg effect which is usually very high in this mutant, is practically reduced to zero. Thus, if in the control (the untreated leaf) the Warburg effect consists in an enhancement of CO 2 -fixation from 270 μmol CO 2 fixed mg·chlorophyll -1 ·h -1 under 21% oxygen partial pressure to 530 μmol CO 2 fixed · mg Chl -1 · h -1 under 3% oxygen, the treated sample yields the maximal rate of photosynthesis already under 21% oxygen partial pressure. At concentrations around 10 -4 м the compound is toxic to the plants and inhibits CO 2 fixation substantially. A second hydroxylamine derivative used in this study has an additional methyl group in the ortho position to the -O-NH 2 group. This compound is already toxic to the plant at concentrations around 10 -5 м at which O-(p-nitrophenyl)-hydroxylamine optimally enhances CO 2 fixation at normal oxygen partial pressure. From our studies it appears that O-(p-nitrophenyl)-hy- droxylamine binds to ribulose-1.5-bisphosphate carboxylase inducing a conformational change of the enzyme. This is concluded from the observation that a monospecific antiserum to ribulose-1.5- bisphosphate carboxylase is exhausted at considerably higher enzyme concentrations when the enzyme has been treated with the hydroxylamine derivative prior to the antiserum addition. The observation is interpreted as being due to a conformational change induced by the compound, leaving less antigenic determinants accessible in the enzyme surface, when compared to the untreated control. The inhibition of photosynthetic CO 2 fixation at high concentrations of both hydroxylamine derivative studied is due to the inhibition of photosynthetic electron transport on the donor side of photosystem II. a phenomenon which is known already for unsubstituted hydroxylamine from the literature. The inhibition of electron transport on the donor side is deduced from an analysis of fluorescence rise kinetics at room temperature.

The aim of this work was to compare the kinetics of platinum binding to “naked” DNA and DNA remaining in a nucleoprotein complex. The incubation of chromatin and DNA from calf thymus was carried out with cw-DDP at different concentrations and incubation times. The coefficient r b /μmolPt/μmolDNA/of platinum binding to DNA was determined and the kinetics of cis-DDP binding to particular preparations was found and compared. The presence of chromosomal proteins decreases the extent of DNA platination. Hence it can be suggested that this drug induces the process of chromatin aggregation and condensation which in turn reduce the release of the total pool of DNA.

Migration of the screening pigment in the compound eye of the sphingid moth Deilephila elpenor is altered by noradrenaline, as shown by microreflectometric measurements on eyes of intact moths and by transmission microscopy on preparations consisting of the screening pigment cells and dioptric structures. Local application of noradrenaline inverts the reaction of the pigment to light stimulation; light causes a contraction of the pigment instead of the normal dispersion. It is suggested that catecholamines are involved in the normal regulation of pigment migration.

(1) In light microscopical studies of living isolated frog irises, it was found that the maximal areas of experimentally light induced contractions in the m. sphincter pupillae were located beneath small illuminated regions. There were no visible contractions of muscle cells outside the illuminated areas. It was shown that exposure to light could directly cause contractions of isolated single sphincter muscle cells. (2) Junctional structures of the iris sphincter cells were studied by means of thin sections and freeze fracture electron microscopy. Intermediate junctions, a few focal tight junctions and occasional small gap junctions were identified. Pit containing intramembranous particles which resemble gap junction connexons were found in large numbers, dispersed over the plasmalemmas of sphincter muscle cells. From these physiological and morphological observations, it is concluded that sphincter muscle cells of the frog iris may be coupled via gap junctions, but that the cell coupling is not sufficiently extensive to form the basis for a functional syncytium.

Using amino-styryl-pyridinium dyes as fluorescent probes we have studied the membrane potential in spheroidal neurons isolated from the circumaesophageal ganglia of the snail Helix pomatia. A spatial resolution of 10 μm is attained at a time resolution of 2.5 ms with a typical signal-to-noise ratio of 10/100 mV as referred to an average sensitivity of the probes of 3%/ 100 mV and to a quantum yield of the detector of 0.13. The linear response of fluorescence, the effect of staining, the variation of sampled area and of illumination are characterized.

Cell cycle progression of G2 fractions (75-80% G2 (4C) cells) from 8 h anaerobically cultured asynchronous hyperdiploid Ehrlich ascites tumor cells strain Karzel, separated by centrifugal elutriation, was studied after reaeration by flow cytometric methods, including the BrdU-H33258 technique and dual parameter measurements. Analyses of the growth kinetics demonstrated that one fraction of the cell population proceeds through a normal cell cycle (2C → 4C) with a generation time of about 20 h. Another portion entered a new cycle (4C → 8C) to form cells with a DNA content up to 8C; mono-, bi- and polynucleate cells could be detected. After 15 h aerobic recultivation of the anaerobically cultured G2 cells, a fraction containing 80-85% with a DNA content of → 4C was separated. On recultivation, these cells pass a 4C → 8C division cycle with a generation time of about 10 h, and a G1 period of less than 4 h.

In the last few years we have shown that atmosferics of different frequencies correlate with the onset of epileptic fits, heart infarcts and with the intensity of an inflammation reaction in rats.

The scent secretions of Lethe marginalis (Satyrid butter­ fly) were investigated by means of GC and GC-MS. The scent substance is found to be γ-decalactone.

Suspension cultured Nicotiana tabacum and Catharanthus roseus cells were labeled with [ 3 H]inositol, the phospholipid fraction extracted and separated by thin layer chromatography. Three different solvent systems and reference compounds were used to assign the different 3 H-labeled species by autoradiography. The ratio of [ 3 H]inositol incorporation into PI, PIP and PIP 2 was found to be 95:4:1; with some preparations a lyso-PI band was obtained which incorporated about a tenth of the label of the PIP band. With Catharanthus roseus cells a very faint band between PI and lyso-PI was detected which could not be assigned to a reference compound.

Crohn’s disease/small RNAs/electrophoresis Twodimensional and bidirectional electrophoretic tech­ niques previously developed for the specific detection of circular viroids and virusoids in plant material were used to analyze preparations of low molecular weight RNA from the granulomatous bowel tissue of patients with Crohn’s disease and from corresponding tissue of healthy controls. A major and two minor RNA species of about 300 nu­ cleotides length were detected in RNA samples from eight Crohn’s disease patients and not in those from three healthy controls. It remains to be established whether these disease-associated RNAs with viroid-like electrophoretic properties play a causative role in Crohn’s disease.

The behaviour of cyclic-3′,5′-AMP phosphodiesterase has been studied in the presence of psoralen, 8-methoxy-psoralen (8-MOP), 4,5′,8-trimethylpsoralen (TMP) (usu­ ally used in PUVA therapy), 4.6,4′-trimethylangelicin (TMA) and khellin recently proposed for the same thera­ peutical use. TMP and TMA exhibit a significant inhibitory effect on cyclic AMP phosphodiesterase; a light inhibition is produced by khellin at rather high concentration.

Examples have been described indicating that it is possible to construct expression vectors carrying chimeric genes that will be expressed in plants at either high or low levels, or will be expressed only in specific organs such as leaves, seeds, tubers, roots or flowers. By constructing genes the products of which are transported into chloroplasts it will be possible to protect such chloroplasts against the action of certain herbicides and perhaps to improve their photosynthetic capacity.