Volume 32, Issue 5-6

Stacking, Relaxation Time, Tryptamine, 5′-Adenosine Monophosphate Complex formation between 5′-adenosine monophosphate (5′-AMP) and tryptamine was in­ vestigated by measuring 13 C chemical shifts and spin-lattice relaxation times. The chemical shift changes observed were attributed to ring current effects originating in the stacking of the two respective aromatic moieties and to puckering changes of the AMP ribose. Differences in the magnitude of the shifts of the aromatic carbons were related to the geometry of the complex and compared with those found for AMP selfassociation. Upon complex formation the relaxation times of especially the tryptamine indole carbons were greatly reduced, this was explained by an in­ crease in the particle size. Small changes found for the AMP carbons in solutions without tryptamine result from AMP selfassociation. Deviations from isotropic motion observed for the non-aromatic moieties are discussed.

Benzo (a) pyrene, added to the nutrient solutions of cell suspension cultures of Chenopodium rubrum, was metabolized to oxygenated derivatives. With increasing time of incubation decreasing amounts of the added 14 C-radioactivity could be extracted with 70% methanol or be isolated as benzo (a) pyrene. The amount of metabolites formed increased with increasing time of incubation. After 48 hours most of the radioactivity was found in the extracted residue. These observations and some other indications give reason to believe that some of the metabolites are linked to proteins or nucleic acids.

Six novel keto carotenoids have been isolated from the stick insect. The structure of the pre­dominant red pigment has been assigned to 3,4,3′,4′-tetradehydro-β,β-carotene-2,2′-dione on the basis of electronic, infrared, and mass spectra. Minor pigments are 3,4-didehydro-β,β-carotene-2,2′-dione, 2′-hydroxy-3,4-didehydro-β,β-caroten-2-one, and 2′-hydroxy-β,β-caroten-2-one. Two other com­pounds, β,β-caroten-2-one and β,β carotene-2,2′-dione, have been tentatively identified. The ad­ ditional occurrence of β,β-caroten-2-ol and β,β-carotene-2,2′-diol has been reported recently. All hydroxylated pigments are mainly present as fatty-acid esters. During hydride reduction of the 3,4-didehydro-2-one compounds hydrogenation of the 3,4-double bond was observed as a side reaction. A hypothetical scheme of carotenoid metabolism in the stick insect is proposed. According to this pathway the keto groups are introduced by a pseudo one-step reaction; the hydroxylated pig­ments are thought to be reduction products of the corresponding ketones. The proposed pathway is fitting more to physiological data than an alternative one based on the hydroxyl →carbonyl sequence.

State and function of the histidine residues of aminoacylase were investigated by photooxidation in the presence of methylene blue and by chemical modification with diethylpyrocarbonate. Complete inactivation of the enzyme was observed after oxidation of 4 histidine residues. From the pH dependence of the photooxidation it becomes evident that the inactivation of the enzyme is not a consequence of the simultaneous oxidation of tryptophan residues. The enzyme is also inctivated by chemical modification of histidine residues with diethylpyrocarbonate. Activity is restored by treatment with hydroxylamine. Zn 2+ -ions which are essential for the activity of amino­ acylase protect the available histidine molecules against photooxidation and attack by diethyl­ pyrocarbonate. It is suggested that histidine is involved in the binding of the essential Zn 2+ -ions.

Renal aminoacylase is inactivated by dialysis against metal complexing agents such as o-phenanthroline. Activity can be restored by addition of zinc ions. A zinc dissociation constant of about 10- 10 м at pH 7.8 is obtained by titration of the enzyme with a metal ion buffer. The reactivity of the SH groups of the enzyme is considerably affected by zinc ions. The enzyme contains two essential zinc ions per molecule.

Phenylacetic acid and p-hydroxyphenylacetic acid are formed upon incubation of photosynthetic membranes from the prokaryotic alga Anacystis nidulans with ʟ-phenylalanine and ʟ-tyrosine, respectively. The corresponding phenylpyruvic acids act as intermediates as shown by trapping them as the stable oximino acids. The first step in this reaction sequence appears to be catalyzed by a thylakoid-bound ʟ-amino acid oxidase. Already existing evidence concerning phenylacetic acid formation at thylakoid membranes of higher plants via an ʟ-amino acid oxidase and the results obtained with A. nidulans give another example of aromatic amino acids between chloroplasts and blue-green algae.

The pyruvate dehydrogenase complex from the photosynthetic bacterium Rhododospirillum rubrum was associated with the membrane fraction both in heterotrophically and photosynthetically grown cells. The complex was separated from the membranes and partially purified by precipitation with MgSO 4 and gelfiltration through Sepharose 4B. The purified complex had a specific activity of 1.5 - 2 μmol/min-mg protein and contained the following partial activities: pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoamide transacetylase (EC 2.3.1.12) and dihydrolipoamide dehydrogenase (EC 1.6.4.3). Contrary to other bacterial pyruvate dehydrogenase complexes, the enzyme complex from R . rubrum revealed no cooperativity between pyruvate binding sites. The kinetic constants [K m ) for the overall reaction were (in mм) : 0.14 (pyruvate), 0.07 (NAD) and 0.025 (coenzyme A). The K m for thiamine pyrophosphate was dependent on the nature and the concentration of the divalent metal ion (Mn or Mg) present in the reaction mixture, the values ranging from 0.5 to 3 μm . NADH was a potent inhibitor (K i = 5 μm) of the enzyme complex and the dihydrolipo­ amide dehydrogenase. The inhibition was competitive with respect to NAD. In addition to its rapid inhibitory effect, NADH also inactivated the enzyme. Cysteine partially protected the enzyme complex against NADH-inactivation. Acetyl-coenzyme A also inhibited the overall reaction (Ki = 40 μм) . The inhibition was dependent on the concentration of coenzyme A, but independent of the concentration of pyruvate. Sugar phosphates, phosphoenolpyruvate, citric acid cycle intermediates and nucleosidephosphates (1 mм) had no pronounced effect on the overall reaction.

Small angle and wide angle powder diffraction diagrams (SAXS and WAXS) have been ob­tained from samples of dry phrenosine cerebrosides prepared from beef brain. Two kinds of lamellae bundles exist within the powder at 23 °C. The first (structure I) with 15% of the molecules having a long period P of 65 Å and a paracrystalline g-value (Hosemann and Bagchi [1962]) of 4.2% consists of orthorhombic subcells similar to C n H 2n + 2 (n ≧ 26) buth with larger lateral dimensions. It has an angle of 60° between the chain direction and the lamellae surface which is parallel to the (201) netplanes of the paraffin-like subcells. The second (structure II) constitutes 22% of these lamellae and 63% with a period of 49 Å and triclinic subcells similar to C 20 H 42 . The angle between the chain direction and lamellae surface is here 45°. The SAXS reflections of structure II are analysed with a bimodal statistic which proves that the 65 Åand 49 Å lamellae are mixed statistically. At 66 °C only a structure III exists with a 52 Å long periodicity. Here all orthorhombic lamellae are built in but now their (301) netplanes are parallel to the lamellae surface. They obviously stabilize the structure instead of the (201)-netplanes which stabilize at 23 °C. A Fourier-analysis of the first 4 SAXS reflections of the relatively undistorted structures II and III projected to the normal of the lamellae surface shows a series of 21 Å wide step functions with a periodicity of 51 or 52 Å. This proves directly a head to head and teil to tail structure. The 21 Å wide heads are attributed to two galactose rings with their amide linkages and oxygens of the fatty acids and sphingosines. Knowing their size (9 Å) one unequivocally can phase the other 6 SAXS reflections which give rise to a gap of some Å’s thick between them. The gap is deeper for structure II because here lamellae of two different thicknesses are mixed. Two further small gaps in the center of the bilayers indicate the ends of the double chain molecules. From the line widths of WAXS it is evident that small paracrystallites (mPC’s) exist with 40 to 100 Å lateral dimensions. From SAXS line widths one concludes that bundles of 4 to 7 bilayers build up macroparacrystallites (MPC’s), where each bilayer consists of 10 to 25 mPC’s. Obviously the galactose rings on the surface of the bilayers produce much larger paracrystalline distortions than the methyl-end-groups in paraffins but much smaller ones than the backfolds in mats of polyethylene single crystals. Consequently 4 to 7 lamellae can be found in each MPC. The galactose rings are the cause of larger lateral distances between the paraffin-like chains and generate distortions cor­responding to a g-value of ~2.6% in the mPC’s. This is explained by the high degree of con­formational variety that the chains adopt which is larger than in synthetic polyethylene. This would further explain why all our attempts to obtain better crystallinity failed.

The electrical conductivity of black phosphatidylserine membranes, in solutions of 100 mм NaCl, 10 mм sodium phosphate (pH 7.1), is strongly increased by the intrinsic proteins (“strongly bound” protein fraction) from human erythrocyte membranes. The magnitude of the conductivity increase is highly dependent on the maximum pH-value pH* used during the preparation of the protein (8.0≦ pH* ≦11.8). For each pH*, membrane conductivity λf and protein concentration c are linked by the equation λf=k·-c s , k and s being functions only of pH*. The value of s varies between 1.0 (pH* 8) and 4.0 (pH* 10). It is assumed that the protein-induced conducting sites, at least for protein pretreatment at pH* ≦ 10, are assembled from four protein subunits. The incorporation of the subunits into the lipid bilayer is supposed to occur either as the final tetramer (pH* 8) or as monomers (pH* 10) and possibly dimers (pH* around 9).

Donor cells, which had incorporated dansylcerebroside in their membranes, could further transfer this glycolipid to monolayers of acceptor cells. The ease of transfer varied among acceptor cells, BHK cells being the best and MDBK cells the poorest acceptors of the cells tested. The process of transfer seemed to be mediated by lipids rather than by proteins of the membranes. The mode of attachment between donors and acceptors, such as classified as loose contact, tight adhesion or binding by lectin, did not significantly influence the extent of glycolipid transfer. However, modification of plasma membranes by infection of acceptor cells with myxoviruses re­sulted in enhancement of glycolipid transfer in some cases. Various factors have been evaluated with respect to dynamics of cellular membranes.

The molecular weight of a thylakoid membrane polypeptide with the apparent molecular weight 11 000 was determined by measurement of the sedimentation velocity, the diffusion and the ef­fective partial specific volume. The molecular weight was found to be 6300 and that of the poly-peptide-dodecyl sulphate micelle was found to be 11 200. The frictional ratio was 1.35. In ad­dition, we determined the binding of dodecyl sulphate onto the polypeptide by equilibrium dialysis. We found that 1 g polypeptide binds 0.77 g sodium dodecyl sulphate which corresponds to 17 molecules dodecyl sulphate bound per polypeptide chain. In the absence of dodecyl sulphate the polypeptide aggregates. The molecular weights of the aggregates are in 0.01 м sodium phosphate buffer pH 7.2 150 000 and in a 1 :1 mixture of 0.01 м phosphate buffer and 96% ethanol 365 000. The frictional ratios were 1.07 and 1.16 respectively which points at a spherical shape. The experimental conditions for the determination of the dodecyl sulphate binding were critically scrutinised.

The recombination of chloroplast coupling factor 1 (CF 1 ) and thylakoid membranes in reconsti­ tution experiments was studied through the fluorescence of paired labels covalently bound to NH 2- groups. It was found that maximum recombination is achieved at a ratio of 1.5 -3,μg CF 1 /μg chlorophyll. The addition of chloroplast lipids to the medium enhances the incorporation of CF 1 into the membranes. The rates of ATP formation of the regenerated but labeled system are decreased to 50% of those found in unlabeled control experiments. This is discussed in context with the previous observation, that labeling of CF 1 at similar concentrations inhibits the ATPase activity of the isolated protein completely. The possible use of double labeling in the study of the physical aspects of the reconstitution of the photophosphorylating system is discussed.

The reconstitution of chloroplast coupling factor 1 (CF 1 ) into thylakoid membranes was investigated by the fluorescence of the covalently attached label fluorescamine. In contrast to a func­tional regeneration of ATP synthesis, a rebinding of CF 1 was observed regardless if the protein was in its native, purified state or had been activated for ATPase activity by heat, dithiothreitol (DTT) or trypsin treatment. The reintegration of CF 1 into the thylakoid membrane was estimated to be almost quantitative. Since the label binds covalently and irreversibly to free NH 2 -groups, its effects on the various functional parameters were investigated. Blocking of NH 2 groups leads to inhibition of ATPase activity in isolated CF 1 . Labeled factor used in a reconstitution experiment on the other hand, does still result in considerable regeneration of ATP formation. Attaching the label to whole thylakoids leads to a differential effect on phosphorylation, which is inhibited, and the H + -uptake which seems largely unaffected. A differential effect is also observed on coupled and uncoupled electron transport. The results are interpreted as evidence for an involvement of free NH 2 -groups in the mechanism of photophosphorylation.

Fluids of the human body with low protein content, i.e. aqueous humour, tears, cerebrospinal fluid, and urine were analyzed by a combination of micro-disc-electrophoresis and electroimmuno-diffusion. By this method both qualitative and quantitative statements about the proteins of these fluids can be established.

Monkey skin fibroblasts were infected with simian virus 40. Cells that exhibited the viral tumor antigen were found to retain the normal morphology of actin filaments up to six days after infection. However when cells were transformed in terms of focus formation they had lost the normal actin morphology.

Base sequences of many transfer RNA (tRNA) species obtained from different sources contain homologous regions. These homologies, which are 6 to 20 nucleotides long, occur both within the same tRNA molecule and between many different tRNA molecules repeatedly. Since it is very unlikely an 80 or so nucleotide long tRNA molecule could have been formed at once, under primordial conditions, we propose that the homologous oligonucleotides found within the tRNA molecules to-day represent the earliest adapter from which tRNA molecules have evolved.

Polysomes of HeLa cells were adsorbed on Poly (U)-Sepharose columns. This adsorption is probably due to poly (A) sequences at 3′ terminus of the messenger. Stepwise elution initially removed ribosomal subunits thereafter mRNA and a set of proteins. These proteins are identical with the main components of the polysomal messenger ribonucleo-protein particles described previously. Thus, this method allows their rapid and easy separation from ribosomal and other proteins.

Cowpea chlorotic mottle virus RNA has been prepared in comparatively high yield (at least 50%) by a modified phenol extraction method. The preparation, which has high biological activity, has been resolved into four components by zonal centrifugation on a 15 - 40% (w/v) sucrose density gradient. The components obtained have been tested for biological activity against whole plants and plant protoplasts. Each of the two largest components RNA 1 and RNA 2 was by itself infective (50 - 90% of the specific infectivity of the whole genome) and produced virus-specific proteins (coat protein and P2) and RNAs (“RNA 3“ and “RNA 4”). Contamination by small proportions « 10 %) of neighbouring RNAs is presumed to be involved in this infectivity. The two smallest components were obtained in an almost pure form.

Using various envelope mutants of Escherichia coli, the relationship between cell surface struc­ture and the Ca 2+ -or Ba 2+ -dependent competence for transfection was investigated. In contrast with rough strains, smooth bacteria treated with Ca 2+ or Ba 2+ were incompetent for the trans­fection by ØA RF. In E. coli K12 D21 derivatives, Ca 2+ -dependent competence remarkably in­ creased by lpsAl mutation and the highest level of competence was attained by further deficiency in glucose units of the LPS. Upon treatment with BaCl 2 , strain D21 and its lpsAl mutant became highly competent for ØA RF. The effect of Ba 2+ was, however, feeble for lpsAl mutants further deficient in heptose units and/or glucose units. Among different LPS mutants of E. coli B, variation of the Ca 2+ -or Ba 2+ -dependent competence was relatively small and even the competence of strain BB12, whose LPS core contained only two KDO units, was nearly equal to that of wild type bacteria. However, the level of cellular competence induced by Ba2+ was not allways parallel to that induced by Ca 2+ . In mutants deficient in outer membrane protein I, either Ca 2+ -or Ba 2+ -dependent competence increased several-fold, whereas in mutants devoid of outer membrane II*, the com­petence decreased considerably. Unlike nucleoside transport, the uptake of DNA was not affected by tsx mutation.

The influence of the insect hormone ecdysterone on the composition of the RNA from the fat bodies of Calliphora larvae was investigated by hybridization experiments. DNA was iodinated in vitro with 125 J. In saturation experiments, total RNA was hybridized to total DNA; under similar conditions, poly (A) RNA was hybridized to unique DNA sequences. RNA isolated from animals treated with ecdysterone exhibits a higher percentage of hybridization than a corresponding control RNA from the same developmental stage, a result even more prominent when poly (A) RNA was hybridized with unique DNA. It is concluded that ecdysterone induces the transcription of new unique DNA sequences.

The spectral sensitivity of the ocelli of Calliphora was determined by means of the electroretino-gram (ER G). Sensitivity is high in the spectral range from 340 to 450 nm ; with still increasing wavelengths it rapidly decreases. Analysis of the shape of the ERG resulting from stimuli of dif­ferent wavelengths, as well as experiments with selective chromatic adaptation, indicate that there is mainly one spectral mechanism contributing to the response.

Carotenoids were extracted from gametophytes of Allomyces arbuscula at different periods during gametogenesis. After separation by thin-layer chromatography γ-carotene, lycopene and β-carotene were identified from their absorp­tion spectra in visible light. No other carotenoids were detected. After a lag period of 30 min following induction of gametogenesis γ-carotene was the only carotenoid present in extracts, followed by traces of β-carotene at 60 min and by lycopene at 90 min. Accumulation of carotenoids continued during the later stages of gametangia development. Analysis of pigments that had been extracted from asexual mitospores showed that these spores contained both γ-carotene and lycopene.

The flavonol glycosides of Carya pecan and Casuarina equisetifolia were isolated and identified. The branches of C. pecan were found to contain azaleatin-3-arabinoside, azaleatin- 3-rutinoside, quercetin-3-glucoside and caryatin-3′- or -4′-glucoside. From the leaves of C. equisetifolia eleven glycosides of kaempferol and quercetin were identified.

In response to fungal inoculation the leaves of Trifolium arvense accumulate the known isoflavonoid phytoalexins, medicarpin, maackiain, sativan, vestitol, and isovestitol. A previously undescribed isoflavan derivative (arvensan; 7,2′-dimethoxy-4′-hydroxyisoflavan) is also produced by this species. The identification and synthesis of arvensan is described.

The phytoalexin medicarpin has been isolated from the fungus-inoculated leaves of 19 Melilotus species.

In pygidial-glands of water beetles (Dytiscidae) 3-indoleacetic acid and phenylacetic acid have been identified by mass-spectroscopical methods and estimated by UV-spectrometry.

Hog cholera virus grown in PK-15 cells and SK-cells was labeled with [ 35 S] methionine and [ 3 H] uridine. At least 3 polypeptides were resolved by polyacrylamide gel electro­ phoresis after disruption of the virus with sodium dodecyl-sulfate. The molecular weights of the structural proteins were determined to be 55000 (p55), 46000 (gp46), and 36000 (p36). The molecular weight of the viral RNA was determined to be about 4 X 10 6 in polyacrylamide-agarose-gel electrophoresis. In sucrose gradients the RNA has a S 20 ,w value of 40 -45S.

The occurrence of fatal spontaneous leukemia in AKR mice could be drastically reduced by passive immunization with goat antibody against isolated murine Friend virus glycoprotein gp71, possessing high group specific reactivity. The success depended on the time of antibody application. The best results were achieved when mothers and babies were inoculated.

The peritrophic membrane of Daphnia pulex, Daphnia magna, and Leptestheria dahalacensis consists of arbitrarily or hexagonal arranged fibrils. Peritrophic membranes of L. dahalacensis show in addition an orthogonal orientation. The development of these patterns by “self assembly”-processes can be excluded.

In the running activity of the cockroach Leucophaea maderae a strong phase response curve is found when using high intensity light pulses (80 000 lx and about 12 hours duration). The phase response curve has an unsymmetric shape: delays are larger than advances. The phase jump lies about 2 hours after subjective midnight.

In crayfish eyes image formation not due to refraction but to total internal and multilayer reflection in an orthogonal mirror system is described. Relevant optical parameters (geometry and refractive indices) in this eye are determined.

A foraging bee approaching a food source visually fixates the food source before it lands on it. During this process of fixation the bee adjusts its rotatory and translatory flight components to fixed values with respect to all six degrees of freedom.