Volume 28, Issue 7-8

The dependence of the PMR spectra of three purine nucleosides (inosine, adenosine and deoxyadenosine) on temperature, concentration and addition of benzene (0.12 molal) has been investigated in aqueous solution. The interaction of benzene with the nucleosides is deduced from the spectra. Enthalpies of self association have been determined as 2.2± 0.1 kcal·mole -1 for dA and I. The corresponding values obtained for hetero association with benzene are 2.7 ± 0.2 kcal·mole -1 for dA and 2.3± 0.2 kcal·mole -1 for I. The formation of edge to face complexes is concluded. Additional evidence for this type of hetero association has been obtained from the concentration and solvent dependence of the nuclear Overhauser enhancements (NOE) of solutions of the nucleosides. The enhancement factors of aqueous solutions of dA and I depend on the concentrations of the nucleosides. In solutions containing I the NOE found previously in DMSO and the NOE observed in D 2 O differ significantly 1 . The results obtained can be explained by a change of the average glycosyl torsion angle of approximately 10° to 15° in the two solvents.

As psoralen and other furocoumarin derivatives, intercalated between two base pairs of native DNA, under irradiation at 365 nm form inter-strand cross-linkings as a consequence of bifunctional addition, the writers have investigated the ability of psoralen to give such bifunctional photo­ additions, too, with nucleic acids with disordered or partilly disordered structure (denatured DNA and r-RNA). On the basis of fluorimetric, light-scattering, viscosimetric measurements and of the renaturation ability of denatured bacterial DNA, certain results have been obtained. In addition to monofunctional photoadditions, psoralen can give bifunctional binding by irradiation at 365 nm both with denatured DNA and with r-RNA. However, when irradiation of denatured DNA in the presence of psoralen was performed in a concentrated solution (0.4%), the formation of bifunctional additions between two different strands was demonstrated by the increase (50%) of molecular weight of denatured DNA. However, when irradiation of denatured DNA was performed in more dilute solutions (0.1%), the bifunctional photoaddition of psoralen took place producing only bi­ functional additions in the same strand, very probably with the formation of loops, as has been shown by the absence of increase of molecular weight of DNA and by the more restricted structure assumed by the macromolecule, revealed by the light-scattering and viscosimetric measurements. The formation of these bifunctional additions was confirmed by the reduced rate of renaturation shown by denatured bacterial DNA after irradiation in the presence of psoralen. In the case of r-RNA, psoralen, when irradiated can form bifunctional additions only in the same strand.

DNA polymerase, disc electrophoresis, template affinity Two sensitive methods are described for detection of DNA dependent DNA polymerase activities in polyacrylamide gels after their fractionation by micro-disc electrophoresis. One technique is based on the increase in fluorescence of the ethidium bromide complex with template polydeoxyribonucleotides brought about by the action of the polymerases. The sensitivity of the previously described technique has been enhanced. Another method, 14 fold as sensitive, uses radioactive precursors in the enzyme assay after electrophoretic separation; washing, slicing and counting allows to evaluate incorporation into acid insoluble polymer, requiring 3* 10" 2 units corresponding to approx. 0.02 /μg of E. coli DNA polymerase I preparation. Differing activities of polymerase species and differing template preferences may be investigated with these techniques.

The radioprotective ability of ʟ-tryptophan, tryptamine, 3-indoleacetic and indole (10 -4 ᴍ), on UV and gamma irradiated DNA (10 -4 ᴍ) is studied. UV radioprotection is accomplished in the following order of efficiency: ʟ-Tryptophan and 3-indoleacetic acid > indole and tryptamine. The corresponding DRF measured as absorbancy loss at 260 nm, were: ʟ-Tryptophan and 3-indoleacetic acid around 3 and tryptamine and indole around l. When absorbancy loss plus changes in the patterns of the absorbancy curve were con­ sidered the DRF was: L-Tryptophan 2.53, 3-indoleacetic acid 1.54, indole 1.22 and tryptamine 0.75, respectively. For gamma radiation damage the order of radioprotection found was: Indole, ʟ-tryptophan and tryptamine > 3-indoleacetic acid. The DRF when compairing the absorbancy loss at 260 nm, were: ʟ-Tryptophan, Indole and tryptamine around 11 and 3-indoleacetic Acid around 4. When evaluating total changes in absorbancy the DRF were: Indole and ʟ-tryptophan around 6.5, tryptamine 3.7 and 3-indoleacetic 2.1. These data were correlated with measurement of the degree of molecular interactions of DNA and protectors, studied by: Spectrophotometry, m.p. determinations, circular dichroism and oriented circular dichroism, viscosity, and rate of dialysis. Also data of fluorescence and phosphorescense were considered. For gamma radioprotection a mechanism based in radical trapping, local radical trapping and partial recovery of radioprotectors is proposed. UV radioprotection is discussed considering: The close interaction of L-tryptophan with DNA which might impede dimer formation, its proton and electron donor ability. A possible (triplet-triplet) energy transfer from excited DNA to indole derivative is discussed on the basis of acetone competition and a dimer excision process considered.

The equilibrium constants for the complex between ʟ-tryptophan and inosine are established by solubility measurements at different temperatures, the order of magnitude of these constants being about 10 3 times bigger than the corresponding one for the formation of guanosine-tryptophan or any other of the nucleoside-tryptophan complexes. The mode of association of these molecules is governed mainly by stacking interactions as it has been suggested from previous PMR results. The differences in magnitude obtained for the equilibrium constants of (I + T) with respect to (G + T) are discussed in terms of differences in solute-solvent and solvent-solvent interactions. Thermodynamic parameters inferred from the equilibrium constants at different temperatures support this suggestion. Infrared results on I-T complex suggest that changes at the ribose moiety occurs and that possible hydrogen bridges are involved in the mechanism of association besides the stacking interactions.

The solvent dependence of the photooxidation of tryptophan and 3,4-benzopyrene in aqueous solutions was studied by quantum yield measurements. When the hydrocarbon is dissolved in aqueous solution of caffeine, the quantum yields indicate a 3,4-benzopyrene photosensitized trypto­ phan oxidation instead of a photocooxidation, which is indicated in aqueous solution of sodium dodecylsulfate. The same photosensitized oxidation as in caffeine solution is observed, when urea (6 ᴍ) is added to the soap solution, while the fluorescence and absorption spectra indicate no change in the solvation state of the hydrocarbon, comparable to the change from hydrophobic solubilization by the detergent to dipole - induced dipole complex solubilization by caffeine. It is concluded that the difference in the reaction pathways is caused by different solvation states of the excited or reacting oxygen. In the discussion of the results it is referred to reactions of inhibitors.

The area/molecule, A, and surface potential, ⊿V , of plastocyanin (pcyan) at an air-water inter­ face varies with pH. At a pH of about 8.6 a maximum value is obtained for A and ⊿V. At a surface pressure of 3 dyn/cm A and ⊿V are 375 Å 2 and 325 mV, respectively. The dipole moment is 2050 milliDebyes. Pcyan and chlorophyll α (chl) form a mixed film (free energy of mixing and interaction is negative). At a concentration of [pcyan] / [chl] ≈ 1 a maximum interaction is observed both for ⊿V and A (at pH 7.8). Irradiation of the mixed film, in a nitrogen atmosphere, results in an increase in A and ⊿V.

The binding between F-actin and myosin has been studied by analyzing the amount of radio-actively labelled (by means of 14 C-NEM) F-actin and myosin in the formed unsoluble actomyosin pellet after centrifugation of the reaction mixture. In the absence of ATP, the amount of actin bound to myosin varies, depending on the amounts of actin and myosin present, between 0.18 mg actin/mg myosin (2 actin units per 1 myosin molecule) and about 2 mg actin/mg myosin (each actin fibril only uncompletely saturated with myosin). In 0.03 ᴍ KCl ATP decreases, if at all, the affinity of actin towards myosin only slightly; but less actin is bound to myosin in the presence of MgATP and in low ionic strength, indicating that myosin is now more densily distributed over fewer actin fibrils leaving the rest of fibrils free. For precipitation of the total amount of myosin (myosin alone is soluble in MgATP) incomplete saturation of myosin with actin suffices; obviously actin promotes filament formation of myosin. The activation of myosin ATPase by actin depends in a similar manner as actin binding does on actin concentration, hence the enzymatic interaction between actin and myosin is accompanied by true actin-myosin binding. An actin-tropomyosin-troponin preparation, whose reduced viscosity is lower than that of F-actin and which consists of about 30% actin, activates myosin ATPase to the same extent as F-actin does. It competes with F-actin for the same binding sites on myosin and can in the presence of MgATP be displaced from myosin by those preparations of F-actin which have a strong tendency to become fully saturated with myosin. The activation of myosin ATPase by actin-tropomyosin-troponin is reduced after tryptic digestion of actin-tropomyosin-troponin, which affects, according to SDS gel electro­ phoresis, mainly two components of troponin with molecular weights of about 32 000 and 25 000, respectively.

The influence of diffusion on the site of biological objects in sections is estimated. In experiments it is shown that the penetration of fluid in the embedding material depends on the fluid, the embedding material, the temperature, and the time. The hardness of the embedding material becomes smaller by the fluid.

α-Mannosidase was found associated with the vacuolar membranes of yeast. The vacuoles were isolated by flotation from osmotically disrupted spheroplasts of Saccharomyces cerevisiae. The enzyme was used as marker for isolating vacuolar membrane fragments directly from whole cells which were mechanically disintegrated. Over 90% of the total α-mannosidase was recovered in the particulate fraction. The enzyme was present in all of the fractions obtained upon differential centrifugation. Density gradient centrifugation in Urografin (5 - 20% w/v) of preparations obtained by differential centrifugation between 20 000 and 50 000 × g did not result in density equilibrium of the membrane. An isolation procedure involving a sedimentation velocity cut in Urografin gradients has, therefore, been worked out.

Polymyxin B (PX) forms complexes with the O-antigenic lipopolysaccharide (LPS) of Salmonella typhimurium which are stable in aqueous 0.14 ᴍ sodium chloride at neutral pH. PX can be quantitatively recovered from the complex by changing the pH from 7 to 1, or by treatment with the cationic detergent cetylpyridinium chloride. This proves that complex formation does not involve covalent linkages, but is due to electrostatic, and possibly hydrophobic interactions. Quantitative investigations of the binding properties of mutant LPS of the chemotype Rc and Re, of degraded polysaccharide and isolated lipid A point to the negatively charged ketodeoxyoctonate-lipid A region of LPS as binding site for PX. These biochemical results are confirmed by a blueshift of the fluorescence emission maximum of LPS-bound mono-Ar-dimethylaminonaphthalenesulfonyl PX from 550 to 515 nm which corresponds to a location of the dansyl-PX at a polar-apolar interface 34 . Using a published molecular weight of the Rc LPS of 10 000 29 , it is calculated that one LPS molecule can bind 2.7 molecules PX. It is discussed that the PX-LPS interaction may be necessary to alter the permeability of the outer membrane which is a prerequisite for the antibiotic to gain access to it’s final target, the cytoplasmic membrane.

Cycloheximide inhibits transport of K through barley roots without affecting K uptake and accumulation in the root cells. The inhibitor acts upon protein synthesis but does not appear to uncouple respiration. Requirement of protein synthesis for ion transport through roots is possibly due to involvement of symplasmic transport or vesicular secretion into the xylem.

The main pigments of red Lonicera berries are Rhodoxanthin and two hitherto unknown ketocarotinoids named Loniceraxanthin and Webbiaxanthin. The structural formula for Loniceraxanthin is established (2) and for Webbiaxanthin a structural formula is discussed (3).

Digitolutein and two new anthraquinones were isolated from the roots of Digitalis viridiflora. The chemical structure of the new anthraquinones (V 7 , V 8 ) was established to be isodirysophanol- 8-methyl-ether and 5-hydroxydigitolutein by IR, NMR and mass spectra and by comparison with synthetic sample (V 7 ) . The drug contains ten minor pigments besides these major pigments.

Within a dark period of 24 hours green Hordeum plants change their lipoquinone and carotenoid metabolism to that of etiolated plants. A longer darkness induces degeneration of chloroplasts with a concomitant destruction of pigments and lipoquinones which is similar to that of the natural chloroplast degeneration in leaves. 1. The chlorophyll and vitamin Kt content is not augmented in the dark, but decreases. The concentration of the oxidized benzoquinones plastoquinone-9 and α-tocoquinone decreases rapidly whereas the level of plastohydroquinone-9 is augmented until 24 hours darkness. α-Tocopherol in turn is accumulated throughout the dark period. 2. Among the carotenoids β-carotene is considerably enriched only in the first 24 hours of the dark period. Xanthophylls are accumulated up the 3 days. The successive increased appearance of single carotenoids during the dark period corresponds to their biogenetic sequence. During carotenoid breakdown (after the 3rd day) neoxanthin, as common oxidation product of all carotenoids appears as the main carotenoid component. 3. It is postulated that the accumulation of the reduced quinone forms (α-tocopherol, plastohydroquinone- 9) in the dark, which has been shown here, also occurs in the dark phase during natural day-night growth of plants and thus gives rise to the continuous accumulation of a-tocopherol and plastohydroquinone-9 in chloroplasts, as described before 4 .

Free and protein-bound amino acids in serum and scales were investigated. In serum the bound amino acids of psoriatics are significantly higher with exception of Pro, Met, Tyr and Phe in contrast to normal subjects. For free amino acids the differences between normal subjects and psoriatics found in serum and scales are not significant. Results are discussed in relation to the single amino acids and the biochemical correlations are outlined which takes the pathological process as a basis.

The uropygial gland wax of the genus Ploceus is found to be mainly one wax consisting of 2,4- dimethyl-heptanoic acid and n-octadecanol. The wax of one species of the genus Quelea investigated, mainly has two components consisting of 2,4,6-trimethyl-nonanoic acid and n-octadecanol as well as n-hexadecanol. The wax of the sparrows (Passerinae) belonging to the same family Ploceidae is quite different from the uropygial gland fat of these genera by the occurrence of 3-methyl fatty acids and 3-methyl-alcanols.

A receptor protein was isolated from the epidermis cells of the pupae of Tenebrio molitor L. The affinity constant of the complex of this receptor protein and a juvenile hormone analogue was found to be 4.4 · 10 10 l/mole. Its molecular weight is about 3.5·10 5 . The isoelectric point is 4.4.

An antagonistic influence of lithium and thiocyanate ions on the metabolism of nucleic acids has been demonstrated in Tetrahymena pyriformis. This effect corresponds to morphogenetic observations on other ciliates. Similar investigations on mammalian cells (L-cells, CV I-cells, Chang-livercells, HeLa-cells, and ascites tumor cells of Walker-carcinosarcoma 256, Yoshida-sarcoma, Zajdelahepatoma) showed, in contrast to the results in Tetrahymena, no antagonistic influence of lithium and thiocyanate ions: Both ions inhibit the DNA-synthesis, in tumor cells as well as in normal cells.

In double label pulse experiments DNA methylation was compared to DNA synthesis after partial hepatectomy. 24 hours after the operation the highest 14 C-thymidine incorporation rates were found as well as the highest 5-methylcytosine labelling derived from ( 3 H-methyl) -methionine. However, synthesis was much more elevated than DNA methylation. Applying Endoxan DNA methylation is reduced to a significantly higher extent than DNA synthesis. Our results indicate that DNA methylation occurs not only combined with DNA synthesis.

Single- and double-stranded (RF) DNA of page ΦX-174 were 60 Co-gamma irradiated. Dependence of radiosensitivity on the concentration of cysteamine and cystamine was measured. Radioresistance of DNA in vivo (phage particles) as well as of DNA in vitro (infectious DNA) increase between 10 -4 and 10 -1 ᴍ of superprotective agents. The same relationship is observed when the DNA is in the dry and in the wet state. By fractionating radiation damaged RFI and radiation induced RFII by means of sucrose gradient sedimentation it was possible to measure the protective effect of cysteamine on lesions other than strand-breaks. A pronounced radioprotective effect of cysteamine on the production of single-strand breaks in RF -DNA was found (DRF 20). The convenience of the RF system for studying effects of radioprotective agents on the occurrence of different radiation induced lesions is discussed.

After uptake of native T 4 phage DNA in seedlings of Matthiola incana this DNA is integrated as a double-stranded fraction with higher density than the unaltered phage DNA and the bulk of plant DNA.