Volume 47, Issue 9-10

The last decade has witnessed steadily growing support for the notion that the carbohydrate portion of glycoconjugates is not merely an inert structural addition to the protein or lipid backbone. Considerable attention has been given to the chemical composition of glycosidic residues in such heteropolymers. Sialic acids are frequently occurring components of oligosaccharide side chains in glycoconjugates of most higher animals and a few microorganisms. They appear to play an important role as ligands in glycobiological interactions. Mediation of a proposed protein-carbohydrate recognition will necessarily involve a binding protein with the respective specificity. Such proteins thus are able to serve as receptors for certain types of carbohydrate moieties like sialic acids in vivo. Various members of this class of proteins have already proven their value as analytical tools in studying expression and localization of defined sialoglycoconjugates. These proteins attract much attention due to both their functions in situ and their potential as laboratory tools in glycoconjugate research in areas like biochemistry or histology. We present a survey of the purification, characterization and application of this class of proteins to illustrate the status of knowledge and the current directions of research in this field.

In Plagiomnium elatum the new di-C-glycosides 6-C-β-D-glucopyranosyl-8-C-α-L-rhamnopyranosyl- luteolin (Elatin) and 6-C-hexosyl-8-C-rhamnosyl-chrysoeriol were detected. Furthermore four known flavone-C-glycosides were identified. The main glycosides of Plagiomnium cuspidatum are 6-C-glucosyl-7-O-glucosides of apigenin, luteolin and chrysoeriol respectively.

Epicuticular leaf waxes of Castanea sativa and Aesculus hippocastanum contain the same lipids in form of homologous series of hydrocarbons, wax esters, aldehydes, primary alcohols and fatty acids in similar concentrations without any main component dominating. In Ae. hippocastanum wax acetates are present, additionally. Both waxes are found to contain triterpenols and triterpenol esters in remarkable amounts. β-Amyrin, α-amyrin and lupeol are present in both plant waxes, in Ae. hippocastanum wax friedelanol and friedelanone, additionally. The epidermis of both plants are covered with a thin continuous wax layer without crystalloids. But the adaxial leaf surface of C. sativa shows granular wax sculptures and therefore a different micromorphological ultrastructure for both leaf sides.

. From Pilotrichella cuspidata the new biflavonoid 3′,3‴ -binaringenin was isolated. Its structure was elucidated spectroscopically.

Nitrate reductase is induced in cells of Haloferax mediterranei by the presence of nitrate upon anaerobic conditions. This enzyme was purified more than 35-fold with a yield of 49%. Densitograms of polyacrylamide gel electrophoresis show the preparation to be 85% purity. The best enzyme preparation has a specific activity of 13.6 U /m g protein. It is the first halophilic nitrate reductase that has been purified near to homogeneity. The purification consists of five steps: an ammonium sulphate precipitation and four successive gel chromatographies with Sepharose CL-4 B, calcium phosphate, DEAE-Sephacel and Sephacryl S-200. An average Mr of 170,000 was estimated by gel chromatography and non-denaturing gel electrophoresis. Effectiveness of electron donors, cofactors and inhibitors are reported. At low salt concentration the halophilic nitrate reductase was inactivated following first-order kinetics. The K m for nitrate depends on salt concentration and shows values in the range from 2.5 to 6.7 mM.

Substrate specificity of tea leaf fatty acid hydroperoxide lyase was systematically investigated using an entire series of ω 6-(5)-hydroperoxy-C 14 -C 24 dienoic and trienoic acids as substrates. Unexpectedly, the hydroperoxides of C 22 but not natural substrates, i.e., those of C 18 , showed the highest reactivities for the lyase. The reactivities of the hydroperoxides of trienoic acids were always four to ten times higher than those of the dienoic acids.

Protein preparations from Arabidopsis thaliana and Brassica rapa catalyzed the formation of O-sinapoyl-ʟ-malate using 1-O-sinapoyl-β-glucose as acyl donor. The enzyme involved, 1-O-sinapoyl-β-glucose :ʟ-malate O-sinapoyltransferase (SMT; EC 2 .3 .1 .-), catalyzes in the cotyledons of the seedlings the key step in the conversion of the seed constituent sinapine (O-sinapoylcholine) to sinapoylmalate by way of the intermediate sinapoylglucose.

Tree seedling flooding tolerance for 22 species was assessed under controlled field conditions. Initial heights under control (freely draining soil, n = 20 per species) and flooded (standing water, depth = 10 cm, n = 20 per species) conditions were measured in March 1990. Survival, height and diameter growth were determined after 120 days. Recovery from flooding effects was assessed in the following growing season from March to August, 1991. Taxodium distichum (L.) L. C. Rich, exhibited enhanced growth when flooded. Acer saccharinum L., Fraxinus excelsior L., and Quercus robur L. increased diameter but not height growth. The following species exhibited reduced growth and/or survival: Acer campestre L., Acer pseudoplatanus L., Acer rubrum L., Betula nigra L., Betula papyrifera Marsh, Betula pubescens Ehrh., Betula pendula Roth, Crataegus monogyna Jacq., Fagus grandifolia Ehrh., Prunus padus L., Prunus serotina Ehrh., Quercus palustris M uenchh., Quercus petraea (Mattuschka) Liebl., Rhamnus cathartica L., Salix purpurea L., Sorbus aucuparia L., Tilia cordata Mill., and Ulmus glabra Huds. emend. Moss. Recovery from flooding in the second growing season was well established with A. saccharinum, C. monogyna, Qu. palustris, Qu. petraea, S. purpurea, U. glabra, while height growth relative to the flooding period was retarded in A. rubrum, F. grandifolia, F. excelsior, Qu. robur, Rh. cathartica, and S. aucuparia. Mortality increased with B. p a pyrifera and F. grandifolia. Flooded trees of B. nigra, B. pendula, and Rh. cathartica appeared to be strongly handicapped by losing their natural resistance against frost even in the following growing season. By decreasing shoot height growth and biomass production, long-term flooding is suggested to reduce the competitive ability of most tree species in the succession of natural forests in habitats which will be inundated more frequently in future when precipitation is increased by the predicted climatic change.

Liquid cultures of Chlorella kessleri, Scenedesmus armatus and Chlamydomonas reinhardtii have been grown phototrophically in weak (0 .2 -2 G) alternating (7.8 Hz) magnetic fields. The data indicate, that the rate of cell division is larger at 0.2 than at 2 G, viz. that the higher fields have inhibitory effects on cell division and that there is possibly an optimum at ≈ 1 G. N o reliable effect is found on the pigment contents.

When young plants of barley and wheat grown in hydroponic culture were subjected to salt stress, their freezing tolerance increased with increasing severity of salt stress. Detached leaves from salt-stressed plants also exhibited an increased ability to supercool. Avoidance of ice formation permitted leaf survival at subzero temperatures which were no longer tolerated when ice nucleation resulted in extracellular freezing. The increased freezing tolerance under salt stress is attributed to osmotic adjustment of the plants. Increased cellular solute concentrations decrease the extent of cellular dehydration at freezing temperatures, thereby decreasing mechanical and chemical stresses on biomembranes during freezing and thawing.

The common soil green algae Chlorella vulgaris and Stichococcus hacillaris increase extracellular abscisic acid (ABA) production under salt, acid or drought stress 5 - 10 times. Production of ABA also increases from young to senescent cultures 2 - 3 times. The relationship between age of cultures and reaction to stress factors was found. Extracellular ABA levels are 1 - 2 times higher than cellular ones. Possible influence of ABA to soil microbial associations, growth of plants and soil fauna is discussed from an ecological point of view.

The accumulation of betacyanin was reduced by the addition of various cytokinins to suspension cultures of Phytolacca americana. The decrease in the accumulation of betacyanin was overcome by exogenously supplied tyrosine which is a precursor of betacyanin. Benzylaminopurine (BAP) decreased the level of free tyrosine in the cells. Feeding experiments using labeled tyrosine revealed that BAP reduced the incorporation of labeled tyrosine into betacyanins (to about 50% of the control rate). These results suggest that both the availability of tyrosine and the biosynthetic activity of the pathway from tyrosine to the betacyanins are involved in the inhibition of the accumulation of betacyanins by cytokinins in Phytolacca americana cells.

We demonstrate here that monohalogenated acetates (MFA , monofluoroacetate; M CA, monochloroacetate; MBA, monobromoacetate) are unique probes of the electron acceptor side of the photosystem II (PS II) reaction center: (1) they differentially inhibit the reoxidation of the reduced primary plastoquinone electron acceptor, Q A - , by the secondary plastoquinone electron acceptor Q B , and increase the equilibrium [Q A - ] in the order: MBA ≳ M CA > MFA; and (2) M CA and MBA rephase the PS II electron acceptor side, a rather unusual effect. This results in flash number dependence of [Q A - ] with maxima at even flashes to change to odd flashes. Furthermore, we demonstrate a correlation between the inhibitory activity of the halogenated acetates with their hydrophobicity (i.e., partition coefficient).

The inhibition of the transthylakoid pH gradient, ΔpH, and of photophosphorylation by the local anesthetic tetracaine was investigated with isolated chloroplasts from Spinacia oleracea L. Tetracaine strongly inhibited ΔpH in the presence of low salt concentrations. In the presence of high salt concentrations, the inhibition of ΔpH was much smaller. This effect of salt concentration was observed only when both, cation and anion were easily membrane permeable. It was concluded that the effect of salts on ΔpH inhibition was excerted on the inside of the thylakoid membrane. The rate of photophosphorylation, Vp, driven by the PS Idependent artificial proton carrier phenazine methosulfate decreased with ΔpH in the presence of both, high and low salt concentrations. In contrast, Vp driven by the endogenous proton pumps of PS II + I-dependent linear electron flow was largely independent of ΔpH changes in the presence of low salt concentration. It appeared that energy coupling during linear electron transport, in contrast to artificially produced PS I-dependent coupling, may be localized to membrane-bound proton domains which are not accessible to the employed indicators of ΔpH. The data were discussed with respect to recent hypotheses on localized energy coupling in chloroplasts.

The insect hormone ecdysterone stimulated dinitrogen fixation, heterocyst formation, cell size and protein synthesis in suspension cultures of the cyanobacterium Nostoc 6720 within 48 h when added to the culture at concentrations between 10 -9 and 10 -7 м. In the presence of the hormone the volume of vegetative cells increased by 6-fold and that of heterocysts by nearly twice as compared to untreated controls. All the observed effects were inhibited by the calcium- calmodulin inhibitor, trifluoperazine. Cholesterol used as a null control had no significant effect.

A generally applicable highly effective purification method for antiviral plant proteins, selective for those with ribosome-inactivating properties, was developed. It involved affinity chromatography on Cibacron Blue Sepharose of the extracted plant sap after acid-ethanol precipitation and finally cation exchange chromatography on Mono S (Pharmacia). After the second chromatography step electrophoretically pure proteins from the four plant species, Chenopodium amaranticolor, Dianthus barbatus, Phytolacca americana and Spinacia oleracea, were obtained. These proteins inhibited infection of test plants with alfalfa mosaic virus and in vitro translation of TMV-RNA. The apparent molecular weights of the purified proteins were determined by SDS polyacrylamide gel electrophoresis as: 29 kDa for C. amaranticolor, 33 kDa for D. barbatus, 29 kDa for P. americana and 36 kDa for S. oleracea. Comparisons of the proteins by indirect ELISA and by Western blotting with polyclonal antisera from rabbits revealed that all four proteins were serologically related but not identical. In addition all four proteins were found to be glycosylated.

Fluorescence in situ hybridization (FISH) has found widespread applications in cytogenetics. So far the standard protocols for probe amplification (and simultaneous labeling) by PCR, nick translation and in situ hybridization involve different buffer systems leading to a number of time consuming washing steps even before hybridization. In this manuscript we show a fast technique of a close combination of DNA probe preparation and in situ hybridization (ISH). This method was applied to metaphase chromosomes from human lymphocytes fixed on slides. Two specific repetitive DNA probes, the pUC 1.77 DNA probe and the DYZ 1 repetitive DNA fraction were used, amplified and labeled in different ways. Additional experiments with total genomic male human DNA as the DNA probe suggest that this method may be extended to a large variety of other probes. Moreover the ISH technique described does not require toxic denaturing agents, such as formamide.

The in vitro interaction of the [6S]- and [6R]-stereoisomers of CHO-THFA with human RBCs was investigated in the (therapeutically comparable) concentration range from 1.0 to 12.5 μg/ml. Both compounds are bound to RBCs with a k RBC ranging from 0.13 to 0.75 for [6S]-CHO-THFA and from 0.06 to 0.33 for [6R]-CHO-THFA, respectively. The interaction of the [6S]-form with RBCs is about two times higher than of the [6R]-form. Incubation of CHO-THFA with RBCs over 24 h showed an accelerated disappearance from the test solution for [6R]-CHO-THFA with a mean t 1/2 of 49.9 h in compare to t 1/2 = 58.2 h for the [6S]-enantiomer. The results indicate that RBCs may play a major role for the pharmacokinetics and metabolism of CHO-THFA and may act as an intravasal depot especially for [6S]-CHOTHFA.

Fourteen derivatives of cinnamic and acrylic acids were evaluated for their ability to modulate chemiluminescence, evoked by neutrophils that had been exposed to luminol and phorbolmyristate- acetate. Compounds with one or two hydroxyl groups on the phenyl ring demonstrated significant inhibition of the chemiluminescence, but this inhibition was diminished by methoxylation. Saturation of the double bond in the aliphatic chain of cinnamic acid at C 6 - enhanced the chemiluminescence to a small degree. All three acrylic acid derivatives demonstrated a marked inhibition of the luminol chemiluminescence, indicating that characteristics of the heterocyclic ring is of utmost importance in this activity.

Using the carrier-ion complex valinomycin-K + , current/voltage (I/U ) characteristics were registered for planar asymmetric lipid bilayers composed on one side of a phospholipid mixture and on the other side of rough mutant lipopolysaccharide. This system resembles the lipid matrix of the outer membrane of Gram-negative bacteria. The evaluation of the current/voltage curves yielded a highly asymmetric electrical potential barrier. The total potential difference between the phospholipid and the lipopolysaccharide was -85 mV, a result which cannot be explained by contributions of Gouy-Chapman potentials alone. The possible contribution of dipole potentials and influences of headgroup effects are discussed. It is shown that the asymmetry of the I/U-characteristic results from the differences of the surface charge densities of the two monolayers but not from those of the states of order of their hydrocarbon chains.

Electron micrographs of light sarcoplasmic vesicles fixed with glutaraldehyde and osmium tetroxide followed by contrasting with uranyl acetate and lead citrate have been evaluated by registering their membrane profiles with a microdensitometer. The asymmetric arrangement of the two layers of the vesicular membrane could be ascertained by demonstrating a ratio of 1.5 for the thickness of the outer versus the inner membrane layer which is in general agreement with the proposed protein structure of the calcium transport enzyme. Treatment of the vesicles with low concentrations of vanadate (0.1 mM) results in a significant lowering of the symmetry ratio by 20% by reducing mainly the thickness of the outer membrane leaflet. Removal of the membrane lipids by treating the vesicles with phospholipase A 2 and bovine serum albumin diminishes the membrane surface by 50% resulting in a significant increase of both the membrane thickness and the asymmetry ratio by 30 and 12% respectively. The vanadate induced reduction of membrane asymmetry is accentuated after delipidation indicating that the membrane lipids are not essential for the asymmetric appearance of the native membrane. The stability of the spherical form of the vesicles to delipidation implies that the transport molecules are conically shaped allowing strong mutual interactions. At a measured height of the molecule of 80 Å in the membrane, the vanadate induced change in symmetry would be brought about by compensatory changes of less than 3 Å of the outer (35 Å) and the inner (25 Å) diameter of the cone.

Analysis of bud exudate of Populus violascens by GC-MS identified 26 phenolic components. The bulk of the exudate was composed of caffeic acid esters.

Analysis by GC-MS identified 30 phenolic components of the bud exudate of P. laurifolia. The majority of the exudate was composed of flavanones, flavanonols and chalcones, with pinobanksin-3-acetate being the major component. The bud exudate resembled that of the Asian Section Tacamahaca poplars P. cathayana and P. szechuanica but was unlike that of the North American Section Tacamahaca poplars, P. balsamifera and P. trichocarpa.

The new (-)-(3 R,4 S)-4-hydroxymellein (6) is isolated from the fungus Septoria (Phaeosphaeria) nodorum Berk, together with the previously reported isomer 5. The results so far obtained in this series of metabolites are discussed in relationship with biosynthetic considerations.

Seven species of Salvia and one species of Trichostema were analyzed for their exudate flavonoids. They were found to exhibit flavonoid aglycones in various numbers and different patterns. Most of these are flavones, in which 6-methoxy-substituted compounds predominate.