Volume 40, Issue 9-10

The total synthesis of 1,2-dipalmitoyloxypropyl-3-(2-ammoniumethyl) phosphinate, the phosphino-analog of phosphatidyl ethanolamine is described. The phosphinate analog of phosphatidyl ethanolamine has been prepared by an Arbusov type of reaction between a diacyl-glycerol bromohydrin and 2-bromo-ethylphosphonic acid dimethyl ester for 48 h at 178 °C, followed by a reaction with aq. ammonia to yield the final product. The product was characterized by elemental analysis, phosphono-phosphorus determinations and IR spectroscopy.

Epicuticular waxes were extracted and analysed from leaves of 7 different high alpine plant species, with 3 species harvested at different altitudes: Salix herbacea (1950 m, 2825 m), Leucanthemopsis alpinum (1950 m, 3050 m), Loiseleuria procumbens (1950 m, 2660 m), Dryas octopetala (2400 m), Ranunculus glacialis (2800 m), Soldanella pusilla (2640 m), Oxyria digyna (2640m). Two main fractions obtained from waxes were investigated: hydrocarbons and wax-esters, -aldehydes plus -acetates. Individual wax compounds accumulated differently in the respective plant species. The gross composition did not indicate a strict altitude-dependent change in wax composition, though some single components like the C-29/C-31 alkanes (Loiseleuria) or the C-22/C-26 fatty acid methyl esters in Salix, changed significantly with increasing elevation of plant habitat. The evergreen leaves of Dryas and of Loiseleuria exhibited exceptionally high amounts of triterpenol esters. The results indicate that the formation of epicuticular wax layers is not based on a special adaptation to altitude or high mountain habitats in general.

By means of differential thermoanalysis, the miscibility of the main polar tetraether lipid of Thermoplasma acidophilum with two ester lipids, dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol, resp., in the presence of excess water was studied. It is shown that with increasing fraction of tetraether lipid in the mixture, the transition range of dipalmitoyl phosphatidylcholine is broadened and the temperature of the maximum heat flow (T m ) is shifted to lower temperatures; furthermore, the enthaply change (ΔH) of the transition declines. Similar results were obtained with mixtures of tetraether lipid with dipalmitoyl phosphatidylglycerol. It is therefore concluded that the main polar tetraether lipid of Thermoplasma acidophilum , which essentially forms monomolecular layers, is able to form stable common phases with bilayer-forming ester lipids. Miscibility of the tetraether lipid with dipalmitoyl phosphatidylglycerol, which are both monovalent anions at neutral pH, is also observed in the presence of high proton or calcium ion concentrations.

After determination of hydrolysis conditions which allow the best recovery of the mycosporine components (cyclohexenone 1 and the amino alcohols 2 and 3), these compounds have been searched into mycelia of several fungi. In some of them, 1 was unambiguously detected, although in minute quantities, whilst the unknown natural 2 and 3 could not be identified. So, two alternative pathways of mycosporine biogenesis are under investigation.

Seven isoflavonoids have been isolated from the seeds of Bolusanthus speciosus (Bolus) Harms. Five of these com pounds were identified as the isoflavones genistein (1), biochanin A (2), orobol (3), pratensein (4) and 3′-O-methylorobol (5). The remaining two compounds were characterised as the novel isoflavone 3′-O-methylpratensein (6) and the new 3-hydroxyisoflavanone bolusanthin (7). Identification and characterisation was by means of spectroscopic techniques (UV, IR, MS and 1 H NMR). NOe irradiation of the C-2 proton of 3′-O-methylpratensein (6) was used to establish unequivocally the oxygenation pattern of the B-ring.

1. The unequivocal chemical composition of sporopollenin is unknown. Ir was often hypothesized that sporopollenin represents a derivative of carotenoids and/or carotenoid esters. Proceeding from this working hypothesis the influence of inhibitors of carotenoid biosynthesis (DPA, nicotine, Sandoz) on the sporopollenin accumulation was studied in order to test whether an intact carotenoid metabolism is involved. 2. Drastic changes in carotenoid spectra as well as in carotenoid patterns in pollen extracts were used as a “marker” for the uptake and transport of the inhibitors into the anther loculus, the site of sporopollenin biosynthesis. Of the three inhibitors of carotenoid metabolism, DPA, nicotine and Sandoz, only Sandoz appeared to influence carotenoid metabolism in the anthers. 3. After application of Sandoz the sporopollenin accumulation in anthers and pollen was only slightly affected. The amount of sporopollenin after treatment is similar to, or possibly even marginally higher than the control, if dry weight is chosen as reference. If the amount is expressed with respect to one pollen grain, the content is very slightly decreased in the case of pollen from treated plants. Therefore, it is concluded that severe interference in carotenoid biosynthesis does not result in a subsequent drastic inhibition of sporopollenin biosynthesis. 4. A significant increase of PAL activity under the influence of Sandoz indicates that effects upon phenylpropanoid metabolism are possible.

After administration of sodium[1 - 14 C]acetate to rhizome or root cuttings of Convallaria majalis L. two of the main cardiac glycosides were isolated and examined for incorporated radioactivity. With a detection limit of 0.0003% of incorporated label it can be assumed, that the subterranean organs of C. majalis are not capable of cardenolide biogenesis de novo. Interconversions on the cardenolide level do not take place, too. as was proved by application of (U - 14 C]convallatoxol and examination of convallatoxin, the main metabolite in leaves. Therefore the cardenolide content of the subterranean parts of C. majalis (0.2%) must have its origin in the leaves, from where the cardiac glycosides are translocated to rhizomes and roots, which only fulfil a storage function in this respect.

The irritant and tumor-promoting constituents of latex of Euphorbia urucalli L. originating from South Africa were isolated. They were identified as irritant ingenane and tiglianc type diterpene esters derived from unsaturated aliphatic acids and acetic acid and the polyfunctional diterpene parent alcohols 4-deoxyphorbol, phorbol and ingenol, respectively. The irritant and tumor-promoting esters of 4-deoxyphorbol are predominant and were fully characterized chemically and biologically. They are positionally isomeric 12,13-acylates, acetates e.g. Euphorbiafactors Ti 1 -Ti 4 . As acyl groups they carry homologous, highly unsaturated aliphatic acids of the general structure CH 3 -(CH 2 ) m -(CH = CH)n-COOH (m = 2,4; n =1,2,3,4,5; N = 2n + m + 2). Corresponding diesters of 4-deoxy-4α-phorbol are also present which are biologically inactive. Comparison of structures and biological activities of 12,13-diesters of 4-deoxyphorbol indicates that - for a distinct total number of C-atoms (N) in the acyl moiety - an increasing number of conjugated double bonds In} may increase the irritant but decrease the tumor-promoting activity. Replacement of the hydroxyl function at C-4 (phorbol-12,13-diesters) by hydrogen (corresponding 4-deoxyphorbol-12,13-diesters) does not essentially alter biological activities. Epimerization of 4-deoxyphorbol-12,13-diesters at C-4 abolishes biological activities. The specific chemical properties demonstrated for the diterpene ester irritants contained in the latex of E. tirucalli and hence in all plant parts may be useful in trials to abolish the potential risk of cancer involved especially in occupational mass production and handling of the plant. Some of the structure activity relations of the Euphorbia factors isolated made them excellent tools in experimental cancer research for the analysis of mechanisms of tumorigenesis.

Norfloxacin, a nalidixic acid analogue, inhibited streptolysin S (SLS) production when added to young streptococcal culture. DNA synthesis was mainly affected, but increment of cell mass. RNA and protein was also significantly reduced in streptococci treated with norfloxacin. In stationary phase cells and in the washed resting bacteria, the toxin production was resistant to the drug. Pretreatment with norfloxacin did not abolish the cellular capacity to produce SLS. Although extracellular SLS was detectable at log phase of streptococcal growth, enhanced production of the toxin occurred upon cessation of coccal multiplication. In contrast to norfloxacin, lower concentration of rifampicin inhibited SLS production, even added at late log or early stationary phase. Roles of growth phase, medium and carrier in induction of SLS production were analyzed as well.

The potency of ʟ-valine as an inhibitor of Zea mays acetohydroxyacid synthase (AHAS) is increased more than 80110-fold on conversion to its N-phthalyl anilide derivative which is active at 2 µᴍ. The ᴅ-valine, α-aminobutyric acid, isoleucine and phenylalanine analogs are 11- to 43-fold less potent, and similar N-phthalyl anilide derivatives of other branched-chain amino acids are essentially inactive. Full potency is retained on replacing the phthalimide moiety of the valine anilide with cyclohexane-1,2-dicarboximide or 1-cyclohexene-1.2-dicarboximide groups and partial activity with 4-cyclohexene-1,2-dicarboximide and methyl- or dimethylmaleimide groups. Inhibition of the enzyme and of root growth by the valine derivatives may result from binding at or near the site involved in feedback control of AHAS by ʟ-valine.

Volatile secretions from Dufour’s glands in three species of Dufourea bees, Dufourea (Halictoides) dentriventris (Nylander). D.(H.) inermis (Nylander) and D. (Dufourea) minuta Lepelletier have been studied by gas chromatography and mass spectrometry. It was found that the secretions are composed of complex blends of straight chain saturated and unsaturated 2- and 3-ketones and scries of hexanoic and octanoic esters. Cephalic secretions from males and females of D.(H.) inermis and D.(D.) minuta contain sex- and species-specific blends of methylcarbinols and corresponding long chain carboxylic esters. Mass spectrometric fragmentation patterns of esters are described.

From workers of the red wood ant Formica rufa L. 68 compounds could be identified by combined gas chromatography-mass spectrometry. Abdomina contain a row of bishomologue saturated and unsaturated uneven numbered hydrocarbons with undecane as the major product. In addition, 13 formic acid esters could be identified among which hexadecyl formate is present in particularly large amounts. Main components of the head extracts are methyl 3-ethyl-4-methyl-pentanoate, 3-ethyl-4-methylpentanol and tricosane.

Mandibular gland secretion of two Cataglyphis species were chemically analyzed by combined gas chromatography mass spectrometry. The exudates produced by C. bombycina consisted of citronellol and geraniol while that of C. nigra contained only geraniol. The reaction of both species to the glandular secretions was alarm and recruitment. While the reaction of C. bombycina was strong, that of C. nigra was relatively mild.

By means of electrophysiological investigations, GC- and GCMS-analysis of gland extracts and GC-analysis with EAG-detection (Z)-9-dodecenyl acetate was proven to be a component of the sex pheromone of female Cnephasia longana moths. (E)-9-dodecenyl acetate acts as a pheromone inhibitor, as shown by field trials.

Although crude extracts of cockroach (Periplaneta amencana) corpora cardiaca have been shown previously to affect the activity of adenylate cyclase and phosphorylase, we demonstrate in the present study for the first time that low concentrations (0.5 to 5 pmol) of the synthetic myoactive peptides. M I and M II, also affect these systems; these myoactive peptides are identical to the hypertrehalosaemic hormones I and II, and cause an increase in the concentration of the second messenger cyclic AMP in the fat body.In addition, both octapeptides activate fat body glycogen phosphorylase and promote breakdown of fat body glycogen. Both peptides increase the levels to haemolymph carbohydrate in a dose-dependent manner.

The time course of the reaction of oxyhemocyanin from the arthropod Orconectes limosus with SCN − and NO 2 − was investigated. After addition of one of these anions to oxyhemocyanin solu­tion an exponential decay of A 340 was found, indicating a removal of oxygen from the active site. The order of reaction with respect to ligand was about 3 for thiocyanate and about 1 for nitrite. On this basis, a possible arrangement of ligands in the active site has been proposed. The final product of reaction with SCN − yielded a mononuclear copper EPR spectrum while the reaction product with NO 2 − gave a characteristic spectrum of two coupled Cu 2+ ions. It was possible to remove the ligands from the active site by dialysis. The reaction patterns showed distinct changes in a narrow pH range, 5.3-6.5. A possible effect of structural alterations of the protein moiety on the course of the reaction of oxyhemocyanin with ligands is suggested.

The ratios of the scoparone O-demethylation products scopoletin to isoscopoletin were deter mined for reconstituted complexes of NADPH-P-450 reductase and each of four P-450 isozymes in a 2:1 molar ratio with a 1:1 mixture of [7-O-methyl- 14 C]-and [6-O-methyl- 14 C]-scoparone as substrate. The two phenobarbital inducible forms P-450 PB-B and P-450 PB-D have a 1:0.8 ± 0.05 scopoletin to isoscopoletin ratio, and the two β-naphthoflavone inducible forms P-450 βNF-B and P-450 βNF/ISF-G have ratios of 1:4.4 ± 0.1 and 1:3.8 ± 0.1, respectively. The scoparone-O-demethylation activities of the reconstituted preformed complexes of the four P-450 isozymes are given.

A series of pigments of the bacteriopheophytin a spectral type have been isolated from ageing cultures of Rhodopseudomonas sphaeroides strain R26. These pigments are formed in varying amounts, and can be most readily analyzed in vivo by their absorption in the 530 nm spectral range. They are enriched in sedimenting cells, but their formation is not affected by light. By chromatographic comparison with authentic pigments and chemical correlation, the following pigments have been identified: bacteriopheophytin a, bacteriopheophorbide a (which is the predominant product), pyrobacteriopheophorbide a and a fourth, very polar bacteriopheophytin a-type product of unknown structure. The major portion of these newly formed pigments is present in the cells in a state, in which the near-infrared absorption band is shifted to longer wavelengths. As shown by low temperature fluorescence spectroscopy, these forms are very similar to bacteriopheophorbide a aggregates.

Limited proteolysis of 124 kdalton oat phytochrome in the P fr form is described which leads to a photoreversible 39 kdalton fragment. Whereas the absorption maximum of the P r form is only slightly shifted (from 667 to 660 nm) no long-wavelength band is observed in the P fr form. A “bleached” (i.e. low absorbing) form appears instead with a broad absorption maximum at 640 nm. This property corresponds with that of a 40.3 kdalton fragment obtained from pea phytochrome by limited proteolysis (Yamamoto and Furuya, Plant and Cell Physiol. 24, 713, 1983). Dark reversion of the bleached form to the P r form is faster (t 1/2 - 101 min) than dark reversion of 118 kdalton or 60 kdalton P fr . Low temperature spectroscopy of the 39 kdalton fragment showed that the intermediates lumi-R and meta-Ra are formed like in intact phytochrome or 60 kdalton or 114/118 kdalton fragments. It is discussed that limited proteolysis removed that part of the peptide chain which is responsible for the interaction with the P fr chromophore but that the site for interaction with the chromophore of P r and the intermediates lumi-R and meta-Ra is still intact.

Changes in oxygen evolution or uptake in illuminated plastids were studied during the early stages of greening in etiolated leaves. An oxygen uptake was observed in etioplasts, which persisted even after the appearance of oxygen evolution. This oxygen uptake was most pronounced in etioplasts or in plastids prepared after redarkening the plants. Incubation of pre-illuminated etioplasts in the presence of NADPH resulted in an inhibition of the uptake in a subsequent illumination. Addition of NADPH had three further consequences: a shift of the chlorophyllide absorbance band to 682 nm, an inhibition of pigment photodestruction and the appearance of a light-induced fluorescence transient at room temperature. Inhibition of pigment photodestruction by NADPH was maximal when the experimental conditions favoured the formation of the P 682 chlorophyllide-protein. It is inferred that the oxygen uptake seen under these conditions is due to pigment photooxidation. Inhibition of both phenomena by NADPH is ascribed to the specific interaction of the nucleotide with the pigment-protein complexes and to the ability of these complexes to undergo the light-induced chlorophyllide microcycle, described by Franck and Inoue (Photobiochem. Photobiophys. 8, 85-96, 1984). In the leaf, NADPH is thought to play the same role.

14 C labeled thylakoid membranes of Vicia faba chloroplasts were separated by one-dimensional electrophoresis in gel cuvettes of different length using a 12 - 18% polyacrylamide gradient gel system containing 0.1% sodiumodecylsulfate and 6 ᴍ urea. The use of gels with a length of 22 cm and 60 cm led to the detection of about 40 and 80 polypeptide bands, respectively, after dye staining and 50 and more than 100 polypeptide bands, respectively, after autoradiography. The conditions used strongly denaturate chlorophyll proteins and avoid the formation of different polypeptide conformations. Polypeptides differing in molecular mass from about 3000 to 70000 were separated in sharp bands.

Under conditions where 2′-deoxycoformycin is enzymatically phosphorylated by wheat shoot phosphotransferase to the 5′-phosphate in 15 - 20% yield, coformycin is a relatively poor substrate, and is phosphorylated only to the extent of ≤ 5%. However, chemical phosphorylation of coformycin by modifications of the Yoshikawa procedure led to isolation of coformycin-5′-phosphate in 20% overall yield. Coformycin-5′-phosphate was characterized by various criteria, including 1 H NMR spectroscopy. Comparison of the spectrum with that of the parent nucleoside indicated that the nucleotide is predominantly, although not exclusively, in the conformation anti about the glycosidic bond. Like 2′-deoxycoformycin-5′-phosphate, coformycin-5′-phosphate was a feeble substrate of snake venom 5′-nucleotidase, and is hydrolyzed, quantitatively, at only 2% the rate for 5′-AMP. With 5′-AMP analogues as substrate, the 5′-phosphates of both coformycin and deoxycoformycin were poor inhibitors of the enzyme, with K i values > 0.3 mᴍ. The 5′-phosphates of both coformycin and deoxycoformycin do not significantly inhibit adenosine deaminase (K i > 0.2 mᴍ), but are potent inhibitors of adenylate deaminase {K i ≤ 10 −9 ᴍ). Neither coformycin nor deoxycoformycin are inhibitors of mammalian purine nucleoside phosphorylase. The stabilities of coformycin, deoxycoformycin, and their 5′-phosphates, have been examined as a function of pH, and nature of the buffer medium. In particular, all exhibit instability in acid and neutral media, but are relatively stable in the vicinity of pH 9. Some biological aspects of the overall results are presented.

The recently developed stereospecific sodium salt glycosylation procedure has been successfully applied to the synthesis of the β-ᴅ-2′-deoxyribofuranosides of benzimidazole, 5,6-dihalogeno benzimidazoles, and some 2-substituted analogues in high yield. The 5,6-dibromo analogue was obtained by bromination of the parent nucleoside. These have all been characterized by spectro­scopic methods, including 1 H NMR, which permitted analyses of their solution conformations and comparison with those of the corresponding ribofuranosides. Some biological aspects, including preliminary results on cytotoxicity and antiviral activity, are briefly considered.

In growing cells, K + and Rb + effluxes followed first order kinetics, took place with slight differences between K + and Rb + and were stimulated at acidic pH values. In uncoupled cells and ATP-depleted cells, K + efflux was higher than Rb + efflux. In ATP-depleted cells, the effluxes were not stimulated at acidic pH values. Two different K + effluxes are proposed: (i) the normal efflux, which may be a K + /H + antiport and does not function in ATP-depleted cells, and (ii) the backward reaction of the uptake system, which can be high in depolarized cells. The role of the normal efflux system in K + content and pH regulation is discussed.

The properties of the sarcoplasmic reticulum membranes isolated from slow-twitch type I soleus and fast-twitch type II psoas muscles of control and thyroxine treated rabbits were comparatively studied. Membrane yield, maximal calcium storing capacity, ATP-supported calcium uptake, calcium-dependent ATPase activity and calcium-dependent phosphoprotein formation were found to be 3-10 fold higher in psoas than in soleus preparations. Membrane yield, calcium-dependent ATPase activity, ATP-supported calcium transport and calcium-dependent phospho­ protein are at least twice enhanced in the membranes from soleus muscles of animals treated for 14-21 days with thyroxine. The corresponding capacities of the membranes from psoas muscles are not further augmented by the same thyroxine treatment. The maximal calcium storing capaci­ ty of the psoas membranes is their sole specific property which is significantly increased. The changes in the properties of the soleus muscles’ sarcoplasmic reticulum membranes are engen­dered by an increase from 5 to 30-50% in the number of type II fibres. Since the calcium transporting properties of the sarcoplasmic reticulum membranes from type II fibres qualitatively differ from those of type I fibres, thyroxine does not only affect quantitative but also qualitative parameters of the muscles’ sarcoplasmic reticulum membrane system.

Pea protoplasts dielectrophoretic coefficients were measured in alternating electric fields of frequency 1 M Hz and voltage 5 V applied between two concentric cylindrical metal electrodes of outer and inner radii 0.24 mm and 1 mm, respectively. They do not vary significantly with solution osmolarity, but show a clear expressed maximum in isotonic conditions; the values in 0.4 ᴍ, 0.5 ᴍ, 0.6 ᴍ and 0.7 ᴍ manitol solutions are (6.5, 10.2, 8.8 and 5.6) × 10 −24 A 2 s 4 /Kg, pectively. The average cell radii in those conditions are 14.2 µm, 14.6 µm, 13.8 µm and 13.6 µm. The radii of cell-to-electrode contacts follow the same dependence on the osmolarity as the dielectrophoretic coefficients; they do not depend on the applied voltages up to 18V. The times of cell approach near to the electrode were too short to be explained by the action only of the dielectrophoretic force; consequently an attractive force appeared at very close approach. These results may be of use in future studies of membrane adhesion and fusion.

Xanthomonas campestris pv. campestris produced in-dole-3-acetic acid when grown in liquid culture in the presence of certain minimum levels of tryptophan.

An elicitor of the lignification response in wheat leaves was isolated from the germ-tube walls of wheat stem rust. The active compound causes metabolic changes typically correlated with the resistance response, i.e. the formation of lignin or lignin-like polymers in affected epidermal and mesophyll cells and the increased activities of enzymes involved in the phenylpropanoid-pathway.

The influence on the water relations of the third developing leaf of (2-chloroethyl)trimethylammoniumchloride, a synthetic growth regulator, applied to the roots of young wheat plants (Triticum aestivum L.) has been investigated. The tissue water potential and the pressure potential were found to be reduced by several bars in comparison to the untreated controls, whereas the osmotic potential remained unchanged. The content of soluble reducing sugars was considerably increased in the cell sap of CCC-treated leaves. With this accumulation, however, the turgor was not maintained. Additionally, CCC-treated leaves showed considerably lower transpiration rates and higher diffusive resistance than the controls. Thus, the application of CCC to the roots causes alterations in the water relations of developing wheat leaves, which resemble those induced by water deficiency.

The oocytes of the cephalobaenid Pentastomid Rail­lietiella aegypti were studied by electron microscopy. A prominent feature of vitellogenic oocytes are numerous spherical, oval and elongated mitochondria. Some of these are shown with desmosome-like structures. The associations between mitochondria and the desmosome-like structures have always been observed to lie freely in the oocyto-plasm; there are no apparent connections with the oocytous plasma membrane. Possible functional roles of these complexes are evaluated.