Volume 41, Issue 3

Shoots of Daphne mezereum synthesized daphnetin (7,8-dihydroxycoumarin) more efficiently from [2- 14 C]umbelliferone (7-hydroxycoumarin) than from [2- 14 C]p-coumaric acid, and [2- 14 C]caffeic acid was more poorly utilized still. These findings do not support the idea of derivation of daphnetin via hydroxylation of the caffeic acid ring at the 2 position, followed by lactone ring formation; instead they are consistent with the concept of daphnetin formation by an additional hydroxylation of umbelliferone at C-8. Umbelliferone was recovered with little l4C dilution from emulsin-hydrolysed extracts of shoots fed labelled umbelliferone, and TLC of extracts from untreated shoots revealed two substances yielding umbelliferone on hydrolysis. Evidence is presented from TLC and HP LC analysis that one of these is skimmin (7-O-β-ᴅ -glucosylumbelliferone), not previously reported from Daphne. The tracer experiments further support the theory that umbelliferone is the general precursor of coumarins bearing two or more hydroxyl functions on the aromatic ring.

By chromatography of the bark extract from Hamamelis virginiana a monogalloylhamamelose was isolated. It is shown by 13 C N M R spectroscopy that this compound is a complex mixture of isomeric 5-galloylhamamelofuranoses, 2′-galloylhamamelofuranoses and 2′-galloylhamamelopyranoses. The same equilibrium of isomers is obtained by partial hydrolysis of hamamelitannin (1). Therefore it is concluded that the monogalloylhamamelose is formed in the bark by hydrolysis of 1 also.

When washed streptococci were incubated in a phosphate buffer containing MgSO 4 and maltose (Bernheimer’s basal medium) and centrifuged, no hemolytic activity was detected in the supernatant. Although incubation of the spent medium with carrier oligonucleotide did not yield active hemolysin, the mixture turned to be significantly hemolytic, upon ethanol precipitation and dehydration. Oxygen stability, sensitivity to trypan blue, absence in the spent medium from strain C203U, as well as chromatographic properties demonstrated that the hemolytic activity was due to streptolysin S-oligonucleotide complex. The latent streptolysin S was detected in the streptococcal culture supernatant as well. These results indicate that, even in the absence of exogenous carrier, streptolysin S is produced extracellularly by hemolytic streptococci but suffers rapid denaturation. and that the carrier oligonucleotide serves as an effector for the toxin peptide to assume active conformation, through noncovalent interaction. Effects of several protein denaturants were investigated on the toxin activation in vitro.

The range of inorganic acid normalities for maximum color formation of the phosphomolybdenum- blue complex (under heating) increases by elevating the ammonium molybdate concentration, and at a ratio of molybdate molarity/acid normality equal to 10, there is maximum color development at any acid normality in the range 1-4 ɴ with either HClO 4 or H 2 SO 4 (or their mixtures). On the basis of these features a revised method is described for the accurate determination of phosphonate-P percent of total-P. on lipid extracts and on TLC bands. The color at the final step, in both cases, is developed under the same conditions of molybdate, HClO 4 and H 2 SO 4 concentrations, thus avoiding possible errors produced by the use of two separate calibration curves.

Photosynthetic O 2 -evolution patterns were determined in cells of Chlorella vulgaris 211 - 11 h, grown under air enriched with 2% CO 2 (High CO 2- cells) and under ordinary air (Low CO 2 -cells). Oxygen evolution in these algae was measured as consequence of short saturating light flashes with the three electrode system according to Schmid and Thibault, Z. Naturforsch. 34c, 414 (1979). It was shown that Low CO 2 -cells had the usual Joliot-Kok pattern of O 2 evolution whereas High CO 2 -cells exhibited a more reduced pattern characterized by the fact that maximal flash yield was observed under the 4th flash and that damping due to misses was more important than in Low CO 2 -cells. Moreover, the amplitudes of the amperometric signals in High CO 2 -cells were consistently lower. The observations clearly speak in favor of a more reduced condition of the S-state system, when the cells are grown under high CO 2 . This was confirmed by the fact that higher concentrations of hydroxylamine had to be added to bring Low CO 2 -cells into the maximally reduced condition namely S -2 , than was the case with High CO 2 -cells. Our observations suggest that the redox condition of the S-state system of photosystem II in Chlorella vulgaris is affected either by changes of CO 2 concentrations during algal growth or that the S-state system is only maintained in the 4 state Kok condition when the enzyme carbonic anhydrase is present.

Unicellular green algae differ from plant leaves in their thioredoxin profile. Besides several thioredoxins of regular size (M r = 12,000), the heat-stable protein fraction of extracts from Scenedesmus obliquus cells contains a large protein of molecular weight M r - 28,000 which is designated thioredoxin ƒ on the basis of typical properties, in particular by its capacity to stimulate spinach chloroplast fructose-bis-phosphatase and, to lower degree, E. coli ribonucleotide reductase. The new thioredoxin was purified to apparent homogeneity by chromatography on DEAE cellulose, Sephadex G-50. CM cellulose, and Blue Sepharose. When tested in homologous enzyme systems, reduced thioredoxin ƒ strongly activated algal fructose-bis-phosphatase, but was inactive towards the cytoplasmic algal ribonucleotide reductase; NADP malate dehydrogenase was also stimulated. The protein is missing in extracts from a chloroplast-free mutant strain, C-2A′, but appears together with other chloroplast components upon illumination. Protein ƒ is therefore the main chloroplast thioredoxin of the green algae, probably corresponding to the smaller leaf chloroplast thioredoxins ƒ and m combined. Algal thioredoxin ƒ appears closely related, however, to the large thioredoxin found in a cyanobacterium, Anabaena sp.

Pigment photobleaching was performed in thylakoid membranes of Hordeum vulgare (wild type, mutant Chlorina f2, Norfluranzon treated seedlings) and in pigment-protein complexes (CP-I and LHCP) isolated from H. vulgare and Chlamydomonas reinhardtii. Multiphasic kinetics were obtained in all of the above cases. Energy transfer towards pigments absorbing at longer wavelength is postulated as a general protection mechanism against photobleaching. This mechanism explains a substantial bleaching of carotenoids and a faster bleaching of chlorophyll aggregates, absorbing at long wavelength. These conclusions were valid for isolated complexes as well as for thylakoid membranes, although membranes were less sensitive to light.

Extracts have been rapidly prepared from spinach leaves, and the activity of fructose-6-phosphate, 2-kinase (Fru6P,2kinase) and fructose-2,6-bisphosphatase (Fru2.6P 2 ase) measured. The enzyme activities do not change during light-dark transitions, but there is an increase of Fru6P,2- kinase and decrease of Fru2,6P 2 ase activity over several hours in the light. This increase of the Fru6P,2kinase: Fru2,6P 2 :ase ratio shows that a previously unrecognized mechanism, which may include protein modification, controls Fru2,6P 2 levels in leaves. It operates as sucrose accumulates in the leaf, and will be involved in regulating the partitioning of photosynthate.

The interaction between two 14 β steroids being still unsynthesized and a model receptor binding site is theoretically studied by a molecular mechanics scheme. Both com pounds containing seven-membered rings in the 17 β position are found to form stable hydrogen bonds to the receptor and can be attributed to be potential inhibitors of the Na + ,K + -ATPase activity. Substantial steroid conformational changes necessary for an efficient receptor binding are calculated to reduce the interaction energy by only 10 kJ mol -1 . Therefore, alterations in steroid structure should be generally taken into consideration in the investigation of steroid-receptor interactions.

The effect of cholesterol enrichment and depletion of rabbit erythrocytes on the activities of membrane-bound enzymes, namely (Na + ,K + )-stimulated ATPase. NAD + ase and acetylcholinesterase was examined. The cholesterol content of erythrocyte membranes has been modified by incubation of intact cells with sonicated egg lecithin/cholesterol vesicles (cholesterol/phospholipid molar ratio approx. 2) and with egg lecithin vesicles for time intervals up to 10 hours. The cholesterol/phospholipid molar ratio (CH /PL) of untreated rabbit red blood cell membranes was 0 .92-0.94. Linear increase (up to CH/PL molar ratio 1.72-1.9) or decrease (up to CH/PL molar ratio 0.27 - 0.43) in cholesterol content of erythrocyte membranes was observed over the 10 hours of incubation with egg lecithin/cholesterol and egg lecithin liposomes respectively. Fusion of liposom es to the m em brane or their attachment to the membrane surface was not a significant factor in the alteration of CH/PL ratio. (N + ,K + )-stimulated ATPase, NAD + ase and acetylcholinesterase activities were measured as a function of membrane cholesterol. The specific activities of all three enzymes were progressively decreased with increase in cholesterol content. Partial reversibility of the inhibitory effect of cholesterol was demonstrated by measurements on cells depleted again after cholesterol enrichment. This was confirmed by the fact that a lowering in cholesterol content evoked an analogous activation of enzymes. The possible implications of physicochemical modifications of bulk and annular lipids of membrane-bound enzymes in the inhibition mechanism are discussed.

Vanadate increases the initial phosphatidylinositolphosphate formation rate as well as the steady state level of the above lipid phosphate when phosphatidylinositol associated with the isolated Ca 2+ transport ATPase is phosphorylated either by the membrane bound endogeneously present phosphatidylinositol-kinase or by exogeneously added lipid kinase. Employing an ultrasonicated mixture of pure phosphatidylinositol and Triton X 100 (without membrane proteins) no vanadate effect can be seen. This vanadate effect is probably not mediated through the lipid kinase activity, but more likely, through conformational changes of the Ca 2+ transport ATPase protein. Such conformational changes would lead to a higher degree of phosphatidylinositol exposed on the Ca 2+ transport ATPase and thus a higher substrate concentration. Consequently, the initial phosphatidylinositolphosphate formation rate and steady state level increase.

Dose-response curves for the ability to increase blood carbohydrates of the cockroach are compared for various naturally occurring neuropeptides from the corpus cardiacum of different insect species. The peptides investigated are the (decapeptides) locust adipokinetic hormone I (AKH I) and stick insect hypertrehalosaem ic factor II. as well as the (octapeptides) cockroach hypertrehalosaemic horm ones I and II (M I and M II), adipokinetic hormone II from the migratory locust (AKH II-L), and the crustacean red pigment-concentrating hormone (RPCH ). The data show clearly that M I and M II display almost identical dose-response curves with a maximal response at about 5 pmol and ED 50 values (the amount of peptide which is needed to produce 50% of the hypertrehalosaemic response) of 1.9 and 1.8 pmol. respectively. The stick insect peptide is remarkably more potent (ED 50 value: 0.9 pmol), but the other decapeptide. AKH I, gives only about a 70% response com pared with M I and M II. as does the octapeptide RPCH. The ED 50 values of those peptides are 5.9 and 4.5 pmol, respectively. Biological activity after injection of AKH II-L, which lacks a proline residue in the molecule, is only measurable at pharmacologically high doses. An attempt is made to relate the observed differences in the doseresponse relationships to the amino acid sequences of the neuropeptides.

A non-thermal influence of low-intensity millimeter-wave radiation on the puffing of giant chromosom es from salivary glands of larvae of the midge A cricotopus lucidus (Diptera, Chironomidae) is reported. The effect is manifested as a strong regression in size of a specific puff that expresses genes for a secretory protein (Fig. 1). While millimeter-wave irradiation leads to an about tenfold increase of the regression probability com pared to controls, simulation of the small microwave-induced temperature increase in the sample does not result in a significant effect, thus confirming the non-thermal nature of the microwave irradiation effect. This finding could be of importance for the understanding of the interaction between microwave radiation and living systems and hence for the establishment of safety standards in that frequency regime.

The chemical composition of the secretions from the anal sacs of three species from the genus Genetta has been analysed by means of gas-liquid chromatography and mass spectrometry. The main constituents of the secretion are free fatty acids, hydrocarbons, mono- and diester waxes, triglycerides, alkane diols, and free alcohols. Composition of the secretion in the three species is fundamentally similar, but there are some remarkable quantitative differences of the components m entioned. Some of the alkane-1,2-diols in these secretions have never been found before in nature.

In order to investigate the availability of laser-induced mass spectrometry for the determination of bacteria, ten strains of the genera Escherichia and Streptococcus were chosen. Gram-negative as well as Gram -positive lysogenic and non-lysogenic. pathogenic and non-pathogenic strains were examined. Giant pulses of a Nd: YAG laser (frequency quadrupled, wave-length 265 nm) were used to vaporize and partly ionize the bacterial material. The time-of-flight mass spectra obtained could be discriminated by using the stepwise discriminant analysis provided by BM DP software package. Two different methods relating centrifugation and washing procedures were used. With the first method a correct classification between 50.0 and 90.9 per cent (S. sanguis) by means of mass spectra of positive ions and a correct classification between 27.1 and 80.4 per cent (S. lactis-25) by means of mass spectra of negative ions was observed. Using the second method a correct classification between 40.7 and 93.3 per cent (5. lactis-25) by means of mass spectra of positive ions and a correct classification between 56.7 and 95.0 per cent by means of mass spectra of negative ions was obtained. Here 5. sanguis with 93.3 and S. lactis-25 with 81.7 per cent were significantly differentiated. In this context it must be em phasized that a correct classification of 27 per cent must be considered statistically significant, if 10 different strains are used.

After accidental observations of unicellular algae trapped in the intermediate layer of Trichoplax, dead yeast cells were offered systematically as a food. Some of them could be found several hours later engulfed by the big tetraploid fibre cells closely beneath the dorsal epithelium of the animal. This thin epithelial sheet not only allows the expulsion of accumulated waste substances by temporal separation of the cell connections but obviously gives also access to food particles collected previously on the dorsal surface by flagellar movements. Thus, the uppermost cell layer of Trichoplax permits the passage of corpuscles of limited size between its cells.

The effect of temperature on the permeation of vitamin C across membranes of DPPC vesicles labelled with the spin label 1,14 has been investigated. It could be shown that at temperatures above 40 °C the permeation rate is diminished considerably despite the fact that, at these temperatures, the spin label can rotate freely (above phase transition temperature) indicating that the membrane is more fluid. The results obtained agree well with previous findings (W. Lohmann and D. Holz. Biophys. Struct. Mech. 10, 197 (1984)) according to which the furanoid ring formed by the side chain of this vitamin opens at these temperatures. The existence of a bicyclic side-chain structure seems to be a prerequisite for the permeation of vitamin C.

Spin label 1,14 studies on DPPC membranes revealed an increased rigidity of the membrane with increasing spin label concentrations. This causes a retardation in permeation rate of Na-ascorbate. At temperatures > 40 °C, that is above the phase transition temperature, the ESR spectrum of a spin labelled DPPC membrane is very similar to a free spin label spectrum and yet the permeation rate of NaASC is reduced considerably. From this it can be concluded that the spin label has caused certain reinforcements between the lipids, an effect which cannot be reversed by temperatures of about 60 °C. Structural and functional investigations of membranes using spin labels have to consider always the effect described.

A homogeneous magnetic field of 1.1 T strength exhibits a significant influence on the activity of the enzyme ascorbic acid oxidase in vitro. A Lineweaver-Burk plot of the reaction shows the typical pattern of a mixed-type inhibition, i.e. a larger rate of reaction at low substrate concentrations and a smaller rate of reaction at high substrate concentration than that of the control without magnetic field applied.

The oxygen atom in the hydroperoxy group of 13-hydroperoxylinoleyl alcohol was primarily incorporated into the carbonyl group of 12-hydroxy-(3Z)-dodecenal but scarcely into n-hexanal during the incubation with tea chloroplasts.

Vulpinic acid has been isolated from sporophores of Pulveroboletus ravenelii, its first reported occurrence in a Basidiomycete. Permethyl ethers of methyl xerocomate and methyl atromentate and its corresponding monochloro derivatives are produced by P. auriflammeus of Japanese origin.

From the defensive secretion of the rove beetles (Coleoptera: Staphylinidae) Ontholestes murinus (L.) and Xantholinus glaber (Norden) nine volatiles have been identified by GC-MS data. From O. murinus a spiroketal was recorded for the first time from an arthropod defensive secretion. X . glaber has been found to represent the first staphylinid sequestering the monoterpenes limonen and isopulegol.

The isolation and identification of the phosphonolipids from beef brain - right lobe, left lobe and cerebellum - is herein reported. The phosphonolipids were isolated by preparative thin layer chromatography (TLC) using the solvent system methanol/water (2:1, v/v) and identified preliminarily by TLC. The isolated phosphonolipids were subjected to silicic acid column chromatographic separation and the thus separated phosphonolipids were identified by TLC, nitrogen-phosphorus determinations and IR spectroscopy.