Band 35, Heft 11-12

The solution conformations of 9-β-ᴅ-arabinofuranosyladenine analogues modified at the 2′,3′ or 5′ positions are derived from the HRNMR spectra and the longitudinal relaxation rates of the protons. The compounds studied are: 9-β-ᴅ-arabinofuranosyladenine and its 2′-amino-2′-deoxy, 2′-chloro-2′-deoxy, 2′-azido-2 / -deoxy, 3′-amino-3′-deoxy, 3′-bromo-3′-deoxy, 3′-chloro-3′- deoxy, 3′-fluoro-3′-deoxy, 3 r -azido-3′-deoxy, 2 , ,3 , -diamino-2′,3′-dideoxy, 2′,5′-diamino-2′,5′-di- deoxy and 2′,5′-diazido-2′,5′-dideoxy analogues. It is derived from the data that the conforma­tional equilibria of the furanoside rings can be described by the two state N ⇔ 5 model of Altona and Sundaralingam. The emphasis in this work is to study systematically the influence of the different chemical modifications upon the conformational equilibria of the nucleosides. For the arabinosides it is found that substitution of the hydroxyl groups at C2′ or C3′ by other atoms or groups always stabilizes the N conformation oi the araoinose ring. The only exception is fluorine, where S is stabilized. The preference for the N state is correlated with an increasing population of the g+ rotamer of the exocyclic 5′-CH 2 OD group. From the relaxation study of 2′-chloro-2′-deoxy- arabinofuranosyladenine the position of the syn ⇔ anti equilibrium of the base was estimated to be predominantly anti. Thus a preference for the anti-N-g + conformation was derived for the arabinosides excluding an intramolecular hydrogen bond between the 2′ and 5′ hydroxyl groups that was found in the solid state. The stabilization of the N conformer in the modified compounds can be qualitatively explained by steric effects.

Proton magnetic resonance studies of 9-β-ᴅ-xylofuranosyladenine (xyloA), its 2′-amino-2′- deoxy, 2′-azido-2′-deoxy, 2′-bromo-2′-deoxy, 3′-thio-3′-deoxy, 3′-amino-3′-deoxy, 3′-azido-3′- deoxy, 3′-chloro-3′-deoxy, 3′-fluoro-3′-deoxy, 3′-O-methyl, 3′,5′-diazido-3′,5′-dideoxy analogues and 9-β-ᴅ-lyxofuranosyladenine (lyxoA) have been carried out to study the effect of chemical modifications at the sugar moiety on the solution conformational equilibria in these classes of nucleosides. Analogously to previous studies the xylose pucker can be described in the two-state N ⇔ S model of Altona and Sundaralingam. For the xylosides, however, a somewhat different N state (C3′-endo-C4′-exo) has to be used than for the ribosides and arabinosides (C2′-exo-C3′- endo). The results of the conformational analysis are that xyloA exists almost exclusively as an N conformed The effect of the substitutions in the modified compounds is to destabilize the N state. This decrease in the mole fraction of N is accompanied by an increase in the population of the g + rotamer of the exocylic 5′-CH 2 OD group. Thus for the xylosides a correlation N-t/g - or S- g + , respectively, can be derived. Proton relaxation rate measurements on 2′-azido-2′-deoxyxylo- furanosyladenine indicate that in the xylosides the standard syn or anti ranges do not represent stable positions for the adenine base, but that a glycosyl torsion angle (Χ∼160°, Υ∼80°) be­ tween syn and anti is preferred. LyxoA behaves similar to the xylosides and also favours the N state of the sugar pucker. In a summarizing discussion the conformational equilibria of the different modified pentosides - ribose, arabinose, xylose and lyxose - are compared. It is shown that generally intramolecular hydrogen bonding does not yield an important contribution to the stabilization of conformational equilibria in solution. It is also not possible to derive a quantitative relationship between such parameters as Van-der-Waals’ radii or electronegativity of the substituents and the mole fractions of the different conformers. It can, however, be seen that in all cases, where the hydroxyl groups at C2′ or C3′ are substituted by a more voluminous atom or group, steric effects become dominant and allow a qualitative explanation of the changes of the conformational equilibria. Only for the smallest and most electronegative substituents, like fluorine, other interactions (e.g. electrostatic) may become important. It is thus suggested that the purine(β)nucleoside conforma­tion is essentially determined by steric interactions between the different parts of the molecule.

A study has been made of the emission spectra at room temperature, in aqueous and alcoholic media, of 4-aminopyrazolo(3,4-d)pyrimidine (APP) and some of its methylated derivatives. The cationic forms APPH + , N 2 -methyl-APPH + and N 7 -methyl-APPH + exhibit intense fluorescence un­der these conditions, the first two exhibiting excitation spectra which differ from the absorption spectra, pointing to the existence of a tautomeric equilibrium in the ground state. From the shape of the excitation spectra, and comparisons with methylated analogues in fixed tautomeric forms, it follows that the emission of APPH + originates exclusively from the species N(2)-H,N(7)-H + , the other forms being non-fluorescent. The proportion of the emitting species, calculated from the ex­citation wavelength dependence of the quantum yield, is in good agreement with data for the ground state. The emission spectrum of APPH + in aqueous medium consists of two bands with λ max 360 nm and 430 nm, which exhibit identical excitation spectra, but are quenched to different extents by H 3 O + . The 430 nm emission band is absent in alcoholic media. A similar behaviour is exhibited by N 7 -methyl-APPH + , whereas the neutral form of this analogue exhibits only the 430 nm band. These results indicate that the long wavelength emission band of APPH + originates from the rare tautomeric species N(7)-H formed in the excited state by photodissociation of the N(2)-H proton from the species N(2)-H,N(7)-H + . This is further confirmed by results obtained with the aid of the basicity method, as well as by salt efects in non-aqueous media. Consideration is given to the possibility of such processes occurring in other analogues of nucleic acid derivatives.

Malate synthase undergoes an X-ray induced aggregation which can be monitored in situ by small-angle X-ray scattering; the analysis of scattering curves, taken at subsequent stages of aggregation, has led to the establishment of a tentative model for an aggregation in two dimensions (Zipper and Durchschlag (1980) Rad. and Environm. Biophys., in press). This model was checked by comparison of appropriate theoretical curves with the experi­mental curves. The theoretical scattering curves for this comparison were obtained by weighted averaging over the scattering curves calculated for various species of hypothetical aggregates. Based on the approximation of the unaggregated enzyme particle by an oblate cylinder, the aggregates were assumed to be composed of 2, 3, 4 or 6 of such cylinders, associated side-by-side in one and later on in two linear rows. The weight fractions of the species were chosen so, that an optimum fit of the experimental mean radii of gyration and mean degrees of aggregation was achieved. The distance distribution functions calculated for the model are very similar to the functions derived from the scattering experiment. Cross-section Guinier plots of the scattering curves of the model reveal the occurrence of one and later on of two pseudo cross-section factors similar to those observed in the experimental scattering curves. The pseudo thickness factor of the model of the unaggregated particle is found to be retained in the model curves for all stages of aggregation. From these results it can be concluded that the model for the aggregation process is essentially consistent with the scattering behaviour of the aggregating enzyme. Small differences between the theoretical and experimental curves may be explained by the idealizations of the model. The comparison of theoretical curves for alternative models, assuming aggregation in three dimen­sions, suggests that these models are unlikely, though small amounts of three-dimensional aggregates cannot be ruled out completely.

The placental transfer of thallium cations in pregnant mice was investigated by determining the thallium concentrations in fetal and maternal tissue 0.5 to 24 h after application of thallium. The maternal dose of thallium was 8 mg/kg body weight throughout. Uterus and fetus were found not to differ from other organs like heart and liver in time course and magnitude of thallium uptake with an initial surge during the first few hours of exposure to thallium and a rapid decrease to steady 12 and 24 h values somewhat lower than those found in the kidney. Diaplacental transfer is therefore assumed comparatively rapid and a specific placental barrier for thallium does not seem to exist. For the determination of thallium concentrations Field Desorption Mass Spectrometry was utilized as a reliable, fast, and sensitive method for the analysis of metal cations in biological material. This method does not require extensive pretreatment of the tissue and total sample amounts in the range of milligrams and less are sufficient for quantitative analysis.

Five sinapic acid esters were isolated from cotyledons of Raphanus sativus and have been iden­tified with the aid of electron impact (El)-, field desorption (FD)-mass spectrometry and NMR spectroscopy: 6,3′-disinapoylsucrose, sinapoylcholine (sinapine), 6-sinapoylglucoraphenine, 1-si- napoylglucose, and sinapoylmalate. Three of these derivatives are metabolically related in the se­quence sinapoylcholine →1 -sinapoylglucose →sinapoylmalate.

High performance liquid chromatography on octyl- and octadecyl-silica (RP-8 and RP-18) stationary phases offers an efficient and rapid method for analysis of sesquiterpene lactones. A mixture of 11 lactones was analyzed using gradient elution with increasing acetonitrile concentra­tion in water and monitoring the column effluent at 210 nm with an UV detector. The described method has been applied to the determination of costunolide in a crude extract from leaves of Laurus nobilis.

A phytoalexin isolated from the fungus-inoculated leaflets of Centrosema haitiense, C. pubescens (3 accessions) and C. virginianum has been identified as 5,7,4′-trihydroxy-6-methoxy-isoflavone (tectorigenin). This compound is variously accompanied by other isoflavones includ­ ing the previously described phytoalexin, cajanin (5,2′,4′-trihydroxy-7-methoxyisoflavone). Sub­ stantial quantities of aesculetin were obtained when C. haitiense leaves were submerged for 48 h in water or fungal spore suspensions. The taxonomic position of Centrosema within the legume tribe Phaseoleae is discussed.

Genistein, 2′-hydroxygenistein, dalbergioidin, kievitone and phaseollidin have been found to accumulate in leaves and stems of Dolichos biflorus (horsegram) following inoculation with the non-pathogens Pseudomonas pisi and Phytophthora megasperma f. sp. glycinea, respectively. They are accompanied by isoferreirin (5,7,4′-trihydroxy-2′-methoxyisoflavanone), a compound not previously reported as a natural product. Coumestrol and psoralidin occur constitutively in Dolichos leaves and stems.

Methods are described for an enzymatic preparation of 14 C-labeled terpenoids. With a cell-free system of a white mutant of Phycomyces blakesleeanus (Mucoraceae) [ 14 C]squalene and [ 14 C- cis]phytoene can be synthesized from [2- 14 C]mevalonate. The application of norflurazon, a phenyl- pyridazinone herbicide, helps to increase the yield of squalene. Furthermore, the liquid endosperm of Echinocystis lobata (Cucurbitaceae) was used for the formation of either [ 14 C(-)]kaurene from [ 14 C]mevalonic acid or [ 14 C-/ra/w]geranylgeranyl pyrophosphate in the presence of Amo 1618. The hydrocarbons formed were purified by alumina-column chromatography and preparative thin-layer chromatography (TLC). Geranylgeranyl pyrophosphate was separated by DE-column chromatography followed by TLC.

A survey of chemical compositions of ten species of Asarum subgenus Heterotropa growing in some islands in Pacific Ocean showed that two groups could be distinguished on the basis of terpene and phenyl propane compositions.

The influence of various factors such as aeration, pH and size of the inoculum on production of axenomycin A, B, and D and on growth of Streptomyces lisandri was studied in batch cultures. An investigation of the nutritional requirements showed that growth and antibiotic production are not necessarily correlated. The yield of acenomycins was increased to 1.7 g per liter medium by repeated selection for a high producing strain. Bioautography showed that these strains produced a hitherto undescribed antibiotic and that all strains tested differed in the total amount of axenomycins produced but not in the composition of the fraction containing antibiotic activity. Addition to the medium of extra amounts of inorganic phosphate and various nitrogen sources showed that both nutritional components selectively inhibited axenomycin formation but did not inhibit growth of Streptomyces lisandri. Good growth of Streptomyces lisandri was observed in the presence of sucrose and its monomers (glucose, fructose), but whereas sucrose inhibited axenomycin formation almost completely, its monomers did not.

Tobacco cells grown photoheterotrophically with high ammonium and sulfate concentrations are able to cope with small amounts of methionine-sulfoximine. The observed methionine-sulfoximine tolerance of tobacco suspensions is due to an extracellular glutathione and an intra­ cellular glutamine reservoir, both reservoirs are considerably reduced by treatment with methionine-sulfoximine concentrations that do not affect the growth of the cells. In tobacco suspension cultures grown with nitrate as sole nitrogen source that do not contain high amounts of glutathione and glutamine, growth inhibition by methionine-sulfoximine can be prevented by addition of these substances to the growth medium. These data indicate that synthesis of glutathione and glutamine are both inhibited by mentionine-sulfoximine; furthermore they show evidence that - in contrary to animal cells -the whole glutathione molecule is taken up by tobacco cells. Synthesis of glutathione from the consisting amino acids is inhibited by methionine-sulfoximine in crude cell homogenates to a similar extent than observed in tobacco suspensions in vivo; therefore, the activity, and not the amount of enzymes of glutathione syn­ thesis seems to be reduced by treatment with methionine-sulfoximine. As tobacco suspensions are able to recover from methionine-sulfoximine treatment with respect to accumulation of glutathione in the medium as well as with respect to growth, detoxication of methionine-sulf­ oximine has to be assumed.

Photoheterotrophic and heterotrophic cell suspension cultures of Nicotiana tabacum have been examined for changes in specific activity of ATP-sulfurylase (EC 2.1.1 A) and OAS-sulfhydrylase (EC 4.2.99.8) during growth on different nitrogen sources. During exponential growth the specific activity of ATP-sulfurylase and OAS-sulfhydrylase remained constant and was on the same level in cells with high and with low rates of sulfate assimilation. The specific activity of both enzymes rapidly increased in green photoheterotrophic cells as well as in chloroplast-free heterotrophic cells after the sulfur from the medium had been used up. This increase was reversed when the cells were transfered back to a sulfate supplemented nutrient solution. The changes in enzyme activity due to sulfur depletion seem to indicate a regulatory mechanism for these enzymes.

5-Aminolevulinate dehydratase (ALAD) from spinach (Spinatia oleracea) was isolated by affinity purification on an immunoabsorbens with a yield of 70 to 80% of the activity in the crude enzyme preparation. The enzyme eluted from the immunoabsorbens was pure as judged by poly­ acrylamide gel electrophoresis and is a hexamer with a subunit molecular weight of about 50000. Enzyme bound to the immunoabsorbens was able to synthesize porphobilinogen in a continuous manner. Owing to the lability of the enzyme and its low abundance in plant tissue, we have been unable to obtain similar yields of purified enzyme using classical purification procedures. This highly efficient purification was made possible by using monoclonal antibodies as described by Köhler and Milstein (Nature 256, 495 (1975)). The availability of monoclonal antibodies meant that it was not necessary to purify the enzyme to homogeneity by classical means in order to raise an antiserum specific for ALAD. Sixteen clones of cells producing anti­ bodies against ALAD were selected. They all expressed a ϰ light chain but differed in the heavy chain class which was either γ 1 or γ 2a . The availability of pure ALAD enzyme and of highly specific antibodies against the enzyme now enables us to answer questions concerning properties, localization, intercellular transport and evolution of ALAD. It is clear that the technique used and the questions asked are not restricted to ALAD.

Following a 20 to 24 h lag-phase after sowing, the onset of both rapid degradation of sinapine (sinapolycholine) and rapid increase in sinapine esterase activity in cotyledons of Raphanus sativus was observed. After 2 days of germination maximal enzyme activity was reached and declined in subsequent germination stages as rapidly as it had appeared. Esterases, active against indophenyl acetate, showed highest activity in dry seeds, declining to more than 50% between the 1st and 3rd day of germination. Starch gel electrophoresis showed that all protein extracts contained a multiplicity of esterases, active against α-naphthyl acetate. When gels were incubated with sinapine, one new band appeared, stainable with diazotized ρ-nitroaniline. This band represents sinapine esterase activity. Tests for substrate specificity towards cinnamic acid choline esters showed highest enzyme activity with sinapine. Studies on the occurrence of sinapine esterase in other Brassicaceae revealed that the enzyme activity coincides with the occurrence and degradation of sinapine.

A glucosyltransferase was isolated from immature “cherry” tomatoes and was partially purified (200-fold) by ammonium sulphate precipitation and successive chromatography on Sephadex G-100 and DEAE-cellulose columns. The enzyme utilised the free hydroxycinnamic acids and UDP-glucose in the formation of their respective glucosides (pH 8.0) and glucose esters (pH 7.0); but did not accept the CoA thiolesters of HCAs in the presence of glucose-1-phosphate. The constant glucoside/glucose ester ratio observed during purification suggests that both reactions are catalysed by the same enzyme. The K m values for ρ-coumaric, caffeic, ferulic and sinapic acids were 0.8, 1.5, 1.4 and 2.5 μᴍ, respectively. With ferulic acid as substrate, the K m value for UDPG was 10 μᴍ. The enzyme required an -SH group for activity and the reaction was strongly inhibited by EDTA, divalent metal ions and UDP.

“Flavanone Synthase” (FS) isolated from oat primary leaves shows maximal activity in the differentiating zone of five day old leaves. Activity of FS is distributed in both the upper and lower epidermis, but dominant in the mesophyll. After fractionation of mesophyll protoplasts of Avena by differential centrifugation, FS activity could only be detected in the “cytosol” and not in intact chloroplasts or other particulate fractions.

The cyanobacterium Synechococcus (Anacystis nidulans strain L 1402-1) was grown at + 37 °C in 3.0 vol.% CO 2 . The effect of preillumination with white light on the subsequent dark 14 CO 2 fixation was studied under aerobic conditions at + 30 °C. The radioactive carbon first incoiporated into 3-phosphoglyceric acid was transferred during the later periods of dark 14 CO 2 fixation to phosphoenolpyruvate and aspartate. No labelling or a very low label in sugar monophosphates could be observed. During the dark/light transients the initial fixation product was mainly aspartate. The pattern of 14 C-incorporation into photosynthetic products under steady state conditions (10 min photosynthesis) varied with the temperature during the experiments. The radioactive carbon was firstly incorporated into 3-phosphoglyceric acid. During the later periods of photosynthetic 14 CO 2 fixation an increased 14 C-incorporation into aspartate and glutamate could be observed. Our findings were interpreted with operating of a phospho­enolpyruvate carboxylation besides the Calvin cycle.

-9-Tetradecenoic acid was isolated from the total fatty acids of beef tallow. (Z)-9-Tetradecenol, (Z)-9-tetradecenyl acetate and (Z)-9-tetradecenal, compounds known to function as sex attractants in various insect species, were prepared from this fatty acid.

Abdominal rinses of calling females of the turnip moth, Agrotis segetum, were analyzed by ca­pillary gas chromatography combined with flame ionization detection, electroantennographic de­tection and mass spectrometry. Traces of a compound showing the retention time and high elec- trophysiological activity of (Z)-5-decenyl acetate could be detected only by electroantennography. In addition, (Z)-7-dodecenyl acetate, (Z)-9-dodecenyl acetate, dodecyl acetate and a compound resembling (E)-5-dodecenyl acetate were found. A mixture of these 5 compounds was as attrac­tive in the field as were virgin females, while (Z)-8-dodecenyl acetate, which was also found in the female, was a strong inhibitor. Wind tunnel and field experiments indicate that (Z)-5-decenyl acetate, (Z)-7-dodecenyl acetate and probably (Z)-9-dodecenyl acetate are essential for attraction.

Electrophysiological analysis of olfactory hair sensilla in male P. pisi has revealed four different types of presumed pheromone receptor cells, maximally responsive to (Z)-11-tetradecenyl acetate (Z11-14:Ac), (Z)-9-tetradecenyl acetate (Z9-14:Ac), (Z)-11-hexadecenyl acetate (Z11-14:Ac) and (Z)-7-dodecenyl acetate (Z7-12: Ac), respectively. These four compounds were tested, singly and in various combinations, for efficacy in attracting P. pisi males in the field. High trap catches were obtained with mixtures of Z11-14: Ac/Z9-14: Ac in the ratio 100/100, whereas the 100/30 and 30/100 mixtures of the two compounds were only slightly attractive. No male P. pisi were captured by single chemicals or binary combinations of Z11-14: Ac/Z11-16: Ac, Z11-14:Ac/Z7-12:Ac, Z9-14:Ac/Z11-16:Ac, Z9-14:Ac/Z7-12:Ac, or Z11-16:Ac/Z7-12:Ac. Various compounds, including Z11-16: Ac and Z7-12:Ac, were tried as third chemicals in addi­tion to 100 μg Z11-14: Ac + 100 μg Z9-14: Ac but none increased trap catches over the basic lure.

The in vivo C-2-hydroxylation of testosterone and 5-dihydrotestosterone in rats was measured by labilisation of tritium as HTO after injection of [1α, 2α-T]testosterone or [1 α, 2α-T]5α-dihy- drotestosterone (radiospirometry). After experimental liver damage caused by CCl 4 or after starvation for 60 h the hyroxylation of the androgens decreased. Similar results were gained by measuring the C-2-hydroxylation of testosterone, 5 α-dihydrotestosterone and estradiol.

The enzymatic activity and quaternary structure of rabbit muscle triose phosphate isomerase remains unchanged in the concentration range from 2 μg/ml to 2 ng/ml. In this concentration range the enzyme can be reactivated after dissociation and denaturation in 6.5 ᴍ guanidine hydrochloride. Removal of the denaturant by dilution and separation of inactive wrong aggregates (5-20%) lead back to active dimers, indistinguishable from the native enzyme as far as enzymatic and physicochemical properties are concerned. Based on the long term stability of the enzyme, the reactivation kinetics were analyzed at low concentrations and 0 °C, conditions where the association of inactive monomers to active dimers is predominant in the process of reactivation. The concentration dependence of the rate of reactivation and the kinetic profiles could be described by a consecutive first-order folding and second-order association reaction scheme with the rate constants k uni = 1.9 × 10 -2 s -1 and k bi = 3 × 10 5 ᴍ -1 · s -1 . This implies that the folded monomers of triose phosphate isomerase, which are intermediate states during reconstitution, cannot possess appreciable enzymatic activity.

The labelling of the protein moiety of the sarcoplasmic calcium transport ATPase by fluo- rescamine suppresses calcium transport, calcium dependent ATPase activity, protein phosphoryla­tion by [γ- 32 P]ATP and [ 32 P]phosphate at different extent of amino group substitution. For the hydrolysis of para nitrophenylphosphate by the calcium transport ATPase it is shown that the relationship between the extent of amino group labelling can considerably be altered by the temperature and the presence of ethyleneglycol. It is shown that the amino residues of the phosphatidylethanolamine moiety do not contribute to the inhibiting effect of fluorescamine labelling. The observations suggest that the different functions of the calcium transport system are based on the cooperation of a varying number of calcium transport ATPase molecules.

Calcium binding sites having low affinity constants of < 10 3 ᴍ -1 were titrated in native sarcoplasmic reticulum vesicles as well as in lipid deprived membranes and in the isolated calcium transport ATPase. Short time calcium binding measurements and the determination of the calcium binding heat allow to distinguish low affinity calcium binding sites located on the external surface of the sarcoplasmic reticulum membranes from those present in the section of the transport molecule directed to the vesicular space. The same number of internal binding sites was found for preparations deprived of their lipid content as well as of preparations depleted of their lipids and of their accessorial proteins. Magnesium interferes with calcium binding to the external as well as to the internal low affinity calcium binding sites. The implications of the existence of the low affinity calcium binding sites in the internal section of the calcium transport ATPase are discussed.

In rat liver slices, a prolonged preincubation resulted in a stimulation of AIB and alanine uptake. The increase of transport of both amino acids can be ascribed to a reduction of K t without an alteration of V t . The enhancement of transport seems to be restricted to the A mediation as indicated by the findings that only the Na + -dependent, MeAIB-inhibitable portion of AIB transport was increased by the prolonged preincubation, and, furthermore, that the uptake of cysteine, a specific substrate for system ASC in liver cells, was not affected. The effect of an extended preincubation on AIB transport was abolished by the addition of high concentrations of AIB and alanine to the preincubation medium. Several other amino acids tested did not affect the increase of AIB transport. The observed stimulation of transport seems not to be due to the derepression of the synthesis of transport proteins, as cycloheximide and 5-azacytidine did not block the increase of AIB uptake. The enhancement of amino acid transport does not appear to be a result of a release from transinhibition since the uptake of alanine was markedly enhanced after 3 h of preincubation while the alanine concentrations in the incubated liver slices did not change significantly.

The maximal binding of antibodies out of monospecific antisera to proteins and lipids onto three different chloroplast preparations is compared. In these preparations different parts of the thylakoid membrane surface are accessible to antibodies. Whereas stroma-freed chloroplasts bind antibodies only at the outer surface, also the inner membrane surface is exposed in the two chloroplast preparations which were obtained by ultrasonication and subsequent fractionating centrifugation. In the ultrasonic sediment the inner surface is prevailing. In the ultrasonic supernatant antibodies do not only react with the inner and outer surface but also with con­siderable parties of the interior of the thylakoid membrane. It was found that the thylakoid membrane surface exposed to the stroma consists preponder­antly of proteins whereas the surface directed towards the interior of the thylakoids consists mainly of lipids. All proteins involved in electron transport such as ferredoxin, ferredoxin- NADP + -reductase, plastocyanin, cytochrome f and the coupling factor of photophosphorylation are detectable in the outer surface. The molecules of the coupling factor span the thylakoid membrane from the outside to the inside. They hinder the binding of antibodies to mono- galactosyl diglyceride and to a polypeptide with the apparent molecular weight 24000. The polypeptide 24000 is a major component of the membrane proteins, and is detected on the outer and inner surface of the membrane. The major part of this polypeptide, however, is located in the interior of the thylakoid membrane. The lipid mixture has a different composition in the outer surface than on the inside face of the membrane. The sulfoquinovosyl diglyceride and the phosphatides, phosphatidyl glycerol, phosphatidyl cholin and phosphatidyl inositol are stronger concentrated in the outer surface than in the inside face. The neutral monogalactosyl diglyceride and di- or trigalactosyl diglyceride, however, occur in the inner surface in higher concentrations than in the outer surface. The major part of the sulfoquinovosyl diglyceride and of the monogalactosyl diglyceride are located in the interior of the membrane.

X-Ray diffraction patterns from oriented purple membrane of Halobacterium halobium show that the packing of proteins within the membrane has a paracrystalline structure. The paracrystalline mean distance fluctuations, g, were found to be 1.5 (± 0.1)% for the (100) packing planes of the proteins. The corresponding paracrystallite sizes are of ~ 850 Å. It is suggested that para­ crystalline lattice distortions within the membrane may be explained by conformational singularities of the proteins or by an irregular packing of adjacent proteins and lipids.

Addition of BrdUrd in combination with prednisolone to HSVi-transformed hamster embryo cells induces an alkaline phosphatase (AP). FdUrd enhances, dThd reduces the inducing capacity of BrdUrd and prednisolone. Induction is prevented by addition of cycloheximide or of cytosine arabinoside. BrdUrd reduces transport and phosphorylation of exogenously applied labeled Urd, especially the amount of UTP, UDP and Urd-diphosphate sugars. The Lineweaver-Burk-plot of uridine-up­take after addition of BrdUrd reveales the characteristics of the mixed type inhibition (competi­tive-noncompetitive).

When reovirus-infected Hela cells are incubated at 43 °C virus-specific messenger RNA is released from the polysomes. It accumulates free in the cytoplasm as messenger ribonucleopro- tein particles (mRNPs). These particles have a sedimentation rate of about 50 S and a buoyant density in CsCl of 1.42 g/cm 3 . Reovirus mRNPs contain, besides all three size classes of reovirus messenger RNA. the same spectrum of proteins found in the polysomal mRNPs from uninfected cells, plus two additional proteins with molecular masses of 70000 d and 110000 d, respectively. Electron microscopic examination of the reovirus mRNP fraction reveals specific Y-shaped structures with a total mean length of 0.5 μm.

O 2 evolution activity of PS II in chloroplast stored in buffer including various supercooling solvents was studied. Viscous solvents such as polyethylene glycol (PEG) was remarkably effective for the increase of lifetime of intact chloroplast. Lifetime of immobilized chloroplast by means of radiation polymerization was prolonged more than 40 days in buffer including PEG 70% (v/v). It was found that viscous solvents having PEG units in molecular chain were effective and methoxypolyethyleneglycol methacrylate monomer (M-23G) was most suitable as a im­mobilization carrier. The immobilized chloroplast with M-23G retained the high activity yield more than 30 days in buffer including PEG.

Two methods used to induce protoplast fusion, involving polyethylene glycol (PEG) and high calcium/high pH, respectively, have been investigated in procedures for the inoculation of isolated tobacco protoplasts with TMV. It has been shown that inoculation in the presence of fusion agents involves two stages. Firstly, virus precipitation on to the protoplast surface and secondly, entrapment of virus particles between protoplasts in zones (putatively) of transient protoplast fusion.

Infectious T lymphocytes were determined in thymuses and spleens of mice acutely infected with LCM virus. Such cells appeared in newborn animals, but their numbers declined when the age at infection increased and none were found in adult mice. Either susceptibility to LCM virus is associated with some degree of immaturity or the probability of murine T lymphocytes to encounter the virus as an infectious entity rather than an immunogen is higher in very young than in immunologically mature mice.

The larvae of Dermestes maculatus are able to undergo several moults in complete absence of the cephalic and prothoracic neuroendocrine system, showing thus spontaneous metamorphosis. Measurements of ecdysteroid contents by radioimmunoassay methods have revealed distinctive larval, prepupal and pupal ecdysteroid peaks, as found in other species where development requires frequent stimulation by the centrally produced hormones. Mass fragmentographic analysis has indicated that more than 80% of the ecdysteroids detected by the radioimmunoassay during the peaks consists of ecdysterone. The prothoracic glands (PG) of D. maculatus larvae are located in the head capsule and in the cervical region. They can be separated from the body by neck-ligation. Only the larval peak of ecdysterone can be correlated with PG function. The prepupal and pupal peaks of ecdysterone are not produced by the central neuroendocrine system. The isolated abdominal fragments of larvae and pupae positively produce ecdysteroids in the course of their independent development, but this synthesis has been subnormal and occasionally delayed in time. In contrast, the isolated thoracic fragments produce well synchronized peaks of ecdysterone and this does not depend on the PG. It thus appears that some thoracic tissue other than PG is able to maintain the physiological concentration of ecdysterone by means of a concentration-dependent feed-back mechanism. Due to spontaneous metamorphosis and extremely good survival we have succeeded to induce in this species: a) premature formation of the prepupal cycle of ecdysterone synthesis in the penultimate larval instar by ligation; b) inhibition and delay of the prepupal peak by means of juvenoid treatments; c) reappearance of the prepupal peak in the inhibited “permanent larvae” by exogenous administration of ecdysterone, or; d) modification of the pupal peak by a qualitatively new pupal-pupal one after juvenoid treatments. These experimental transformations of the ecdysterone peaks have suggested that the kind and nature of the peaks are closely related to nature and stage of the ontogenetic development. We therefore believe that the process of larval-pupal reprogramming has occurred in this species long before the increase of ecdysterone titer in the body. Both the prepupal and pupal peaks do not represent a cause but they show to be rather consequences of the developmental programming. Further features associated with possible physiological role of ecdysteroids in insect development have been briefly discussed.

Human red blood cells suspended in a slightly hypotonic solution of low electric conductivity were exposed to an inhomogeneous and alternating electric field (sine wave, 30 V peak-to-peak value, electrode distance 120 μm, 0.5 to 2 MHz). Due to the dielectrophoretic effect the cells align parallel to the field lines under the formation of pearl chains. At high voltages (10 V amplitude) membrane fusion is observed between the adhered red blood cells in the pearl chains, whereby the chains become attached to the electrodes. In contrast to the pearl chains observed at voltages of up to 5 V amplitude the resulting fused and uniform aggregates which exhibit no recognisable individual cells under the light microscope, remain stable, even after the alternating electric field has been switched off or after haemolysis in response to osmotic shock. The fused aggregates are highly elastic. If the field strength of the applied alternating electric field is further increased they are stretched in the direction of the opposite electrode. Frequently, bridges are formed between the two electrodes. The uniform bridges remain stable for some time even in the absence of an electric field. The possibility of cell fusion and its initiation by electrical breakdown of the cell membranes are discussed.

A mechanism is proposed for pattern formation in developmental subfields. In application to imaginal disks, the compartmentalization appears as a prerequisite for the generation of positional information in the proximo-distal dimension. Cooperation of three or four compartments in the production of a morphogen leads to a cone-shaped morphogen distribution, since a high production rate of the morphogen is possible only at the intersection of compartment borders. The local concentration of the cone-shaped distribution is a measure for the distance from this center and can be used as positional information. In agreement with the experimental observations, the model predicts that (i) the distalmost structures are formed at the intersection of compartment borders; (ii) distal transformation occurs whenever cells of all compartments come close to each other; (iii) distal transformation does not require a complete set of circumferential structures; (iv) mutants exist in which the positional information and not the response of the cells is altered and (v) no distal to proximal intercalation of missing leg segments occur. Regeneration and formation of supernumerary insect legs can be explained as well. Simple molecular reaction can account for this type of pattern formation. The “complete circle rule” of French, Bryant and Bryant (Science 193,969-981 (1976)) for distal transformation may be simplified by a “cooperation of compartments” rule.

After treatment with Alcian blue at high temperatures, nuclei from chicken blood smears showed an intense stai­ning rection, which proved to be dependent on the DNA content of chromatin. The possibility that interactions bet­ween the phthalocyanine chromophore and unpaired bases accounts for this temperature-dependent staining of chro­matin is briefly discussed.

The oocytes of the marine goby Pomatoschistus minutus were analyzed for their amino acid content. Most of the amino acids exist as protein, only a little part is free or peptide-bound. Among the protein-bound amino acids, high levels of glutamic acid, proline, alanine, aspartic acid, valine and leucine were detected. These represent more than 60% of the protein amino acids. Among the free acids, glutamic acid, serine and alanine, are dominant. There are no certain proofs of the occurrence of peptide pools in the oocytes of Pomatoschistus minutus.

Hops are told to promote sleep; manyfold efforts to detect the soporific principle have been unsuccessful so far. Preliminary pharmacological tests lead to the con­clusion that the soporific activity of the exhalation of hops can be explained by its content of 2-methyl-3-butene-2-ol (1) in the volatile fraction. It was found that 1, when given to mices i.p. (0.80 g/kg) produces narcosis for about 8 h; no abnormal behaviour was observed there upon. Due to its water-solubility the concentration of 1 in the essential oil obtained by steam-destination is low; contrary to that, 1, is enriched in the more volatile fraction of hops.

ʟ-usnic acid inactivates urease by formation of high molecular weight aggregates which can reached by a maximum of 880 000. ʟ-cysteine partially reverses the inactivation by stimulating the appearance of active high molecular weight polymers. The existence of two class of binding points for ʟ-usnic acid on the urease molecule is proposed, the first showing high affinity for the ligand, related with the loss of activity, and the second, of low affinity, related to polymerization process.

A comparison of the EcoRI restriction endonuclease digest fragments from daffodil chromoplast and chloroplast DNAs on agarose gels has yielded identical results. The sum of the fragments for each DNA gives a minim um molecular weight of 98.6 × 10 6 .

The water plant Lemna perpusilla has been incubated with the E. coli plasmids pMB9 and pBR325, respectively. Uptake of plasmids has been shown by subsequent trans­ formation of E. coli cells to tetracycline resistance after treatment with Lemna DNA from plasmid-incubated plants. In 7 out of 15 assays we found stable transformants. From the transformation rate an amount of 10 -4 to 10 -6 μg plasmid DNA per 10 μg of plant DNA can be calculated.

Measurements of particle densities on the two freeze-fracture faces of digestive and perialgal vacuole membranes in green and aposym biotic Paramecium bursaria Ehrbg. show distinct differences between the P-faces of both membrane types. The distribution of particle densities is more homogenous on the P-faces of perialgal vacuole membranes than on the P-faces of digestive vacuole membranes. Possibly homogeneity among peri­ algal vacuole membranes reflects the stability of perialgal vacuoles during their life cycle. Perhaps lysosomes cannot fuse with them.

Rotation of budding yeast cells in an alternating non-uniform electric field of low frequency was investigated. Rotation frequency was found to be proportional to field strength above a threshold, and varied from cell to cell. The threshold is inversely correlated with the moment of inertia of the cells, while the slope of rotation frequency versus field strength increases with the moment. Rotation frequencies varied between 1 and 10 cycles per second. Clear differences between the dielectrophoretic behaviour of living and heat-inactivated yeast cells were observed.

Homing foragers of the honey bee (Apis mellifera carnica) were tested for discrimination of colour marks presen­ted to them at the hive entrance. At a low rate of natural il­lumination the bees were not able to distinguish the coloured (yellow, blue) marks from those with a corresponding gray. However, their general orientation was not disturbed. From this the conclusion is drawn that at low light intensities bees have an achromatic vision besides their known co­ lour vision at higher intensities.

Evidence for an immunogenic allatotropic factor in the brain of Locusta migratoria is presented. Antibodies to brain material from mature females were raised in rabbits. In vivo injection of these antibodies into young locusts in­hibited ovary development and resulted in hypertrophic as well as necrotic corpora allata. Antibodies to brains without neurosecretory material were not able to block allatotropic or gonadotropic activity.

In developing clawed toads Xenopus laevis, the compensation mechanism of behavioural defects caused by unilateral labyrinthectomy consists of two components, a vestibular one and a hormonal one. Thyroxine accelerates this compensation.

The relation between the amount of the static vestibulo-ocular reflex arc and the developmental stage is characterized by an optimum function. The reflexes occur before the labyrinth is completely developed, but at this time, bilateral connections of the labyrinth with the oculomotor centers are efficient.