Band 31, Heft 11-12

A small-angle-scattering camera of medium resolution without slithigh-error has been constructed and samples of biological materials as natural fats, murein and lipopolysaccharide preparates of gramnegative bacteria have been investigated.

A spectroscopic study of the keto-enol equilibrium of phenylpyruvic acid (PPA) was performed in water-dimethylsulphoxide (DMSO) mixtures, throughout the composition range and in an ex­ tended temperature range. The thermodynamic quantities of the tautomerism were also calculated. The high value of enolization entropy in pure DMSO is attributed to hydrogen bonding of PPA to this solvent. The experimental conditions and the optimum composition range of the H 2 O-DMSO mixtures for PPA analytical determination were determined.

Two major carotenoids of the stick insect were reinvestigated and shown to be β,β-caroten-2-ol and β,β-carotene-2,2′-diol and not isocryptoxanthin (β,β-caroten-4-ol) and isozeaxanthin (β,β-carotene-4,4′-diol). Both pigments are esterified with fatty acids. The identification is based on co-chromatography with authentic 2-, 3-and 4-isomers of the mono-and dihydroxy pigment, mass spectra, and chemical behaviour. In acid solution β-β-carotene-2,2′-diol is specifically dehydro­ genated and rearranged to ketones with retro structures in analogy to the reaction of β,β-caroten-2-ol as recently reported. The final product of the diol is 4,5-dihydro-4,5′-retro-β,β-carotene-2,2′-dione. This is the first demonstration of β,β-carotene-2,2′-diol in an animal.

The carotenoid biosynthesis of the green alga Ankistrodesmus braunii is blocked if these cells are cultured in presence of sublethal doses of pyridazinone herbicides (San 9789, San 6706, BASF 44521) or of the herbicide difunone (EMD-IT 5914). The amount of colored carotenoids normally found in these algae is reduced drastically and the precursors phytoene and phytofluene are accumulated. Furthermore a decrease in the chlorophyll level occurs in the treated cells, but there is a stronger loss of chlorophyll a, resulting in a lowering of the chlorophyll a/b ratio with time. Concerning the activity of substituted pyridazinones leading to inhibition of carotenoid biosynthesis this effect can be related to the chemical structure of these compounds: a trifluoromethyl substitution of the phenyl ring and a mono- or dimethyl substitution of the amine (San 9789, San 6706) or a methoxy group instead of the substituted amine (BASF 44521) are required both for this effect. Other pyridazinone derivatives with either a trifluoromethyl substitution of the phenyl ring (San 9774) or a dimethyl substitution of the amine (San 9785) or a methoxy group (BASF 13761) are without any effect on the pigment pattern of these algae.

Ethidiumbromide (1) has two amino groups in 2-and 7-position which are protonated in acidic water solution. Both pK a -values of 1 are determined at 20 °C by means of the pH-dependence of the electronic spectra using a iterative calculating procedure, pK a1 = 0.713, pK a2 = 2.43. Acetylation of 1 and quantum mechanical calculations lead to the conclusion that the electronic density at the 7-amino group is greater than in 2-position. Therefore with decreasing pH preferably the 7-amino group is protonated (pK a2 ). followed by the protonation of the 2-amino group (pK a1 ). The pK a of 7-amino-9-phenyl-10-ethyl-phenanthridinium-bromide in water solution at 20 °C is determined to pK a = 1 .2 5 .

Diphosphonates, Dissociation Constants The dissociation constants of ethane-l-hydroxy-1,1-disphosphonate [EHDP] and dichloro-methylene-diphosphonate [Cl 2 MDP] for H + , Ca 2+ , Mg 2+ and Zn 2+ have been determined from pH-titration curves. EHDP forms more stable chelates with Ca 2+ , Mg 2+ and Zn 2+ than Cl 2 MDP. Cl 2 MDP is a stronger acid than EHDP.

The electron spin resonance (ESR) spectra of 15 NO- and 14 NO-ligated Hb Kansas have been measured at 77 K in the range of pH 5 to 10. At low pH the ESR spectrum is the composite of a type I and a type II spectrum which changes to another composite of a type I and type II spectrum at high pH. For the definition of type I and type II spectra and the correlation of these types with two tertiary conformation states see Overkamp et al., Z. Naturforsch. 31 c , 524 [1976]. Both, the type I and the type II spectra observed at low and high pH respectively are different with regard to g-tensors and hyperfine-splitting constants. Therefore at intermediate pH values the ESR spectra of NO-Hb Kansas are the composites of four spectral components. The assignments of the four spectral components to the a and the β chains are arrived at from the comparison of the ESR spectra of the α 2 Mmet β 2 NO and of the α 2 MNO β 2 NO species of Hb M Iwate. α and β chains are both characterized by a pH-dependent spectral transition from a type I to a type II spectrum. The chains are non-equivalent with regard to both the type I and the type II spectra. The type I spectra assigned to the a and the β chains are characterized by g * zz = 2.009 5 with a hyperfine splitting of a * zz ( 15 NO) = 2.36 mT and g zz = 2.008 5 with a hyperfine splitting of a * zz ( 15 NO) = 2.41 mT respectively. The type II spectra assigned to the α and the β chains are characterized by g * ′ zz = 2.005 and a hyperfine splitting of a * ′ zz ( 15 NO) = 3.07 mT and g′ zz =2.005 and a hyperfine splitting of a′ zz ( 15 NO) = 3.31 mT. The change of the hyperfine splitting at g zz during the transition from type I to type II corresponds to an increase of the spin density at the NO by about 25% in both types of chains. Comparison of type I spectra of the NO-ligated α and β chains respectively demonstrates that the spin density at the NO is larger in the β chains than in the oc chains. The spectral types are correlated with functional states defined by the kinetics of NO-binding. Binding of inositol hexaphosphate has no influence on the ESR spectra in the whole range of pH as it is expected if NO-Hb Kansas is in the quaternary T structure.

The catalytic properties of peptides containing histidine, cysteine and aspartic acid in ester hydrolysis were studied. Saturation kinetics were found for the reaction of p-nitrophenyl acetate (NPA) with Z-His-Ala-Asp-Gly-Cys-NH 2 and Z-His-Ala-Gly-Gly-Cys-NH 2 . The Brönsted equation for the hydrolysis of tert-butyloxycarbonyl-L-alanine-p-nitrophenylester (Boc-Ala-ONp) catalyzed by simple imidazole and SH-compounds was determined. The catalytic behaviour of the peptides in ester hydrolysis could not be described by the Brönsted equations for imidazole or thiole catalyzed hydrolysis of NPA and Boc-Ala-ONp. The pH dependence of the rate constants of the catalyzed ester hydrolysis gave no linear plots in 1/k versus H + diagrams.

The photodynamic deactivation of lysozyme in presence of acridine orange is caused by a reaction between singlet oxygen formed via the dye triplet state and the protein. In order to identify the region where the singlet oxygen reacts with the protein we have investigated the kinetics of the deactivation in presence of the inhibitor of the enzymatic reaction N-acetylglucosamine (GlcNAc). The overall experimental rate constant becomes slower with increasing saccharide concentrations. As we can exclude experimentally that this kinetical effect is caused in presence of the saccharide by a physical quenching of singlet oxygen or of the dye triplet state it has to be assumed that GlcNAc protects the surrounding of its binding place at subsite C of the enzymatic center sterically against an attack of singlet oxygen. In this region three tryptophan residues are located, which could be sensitive against singlet oxygen. Surprisingly, however, it has been found that only those species are protected, in which a second saccharide molecule is bound to the protein, probably at subsite E at the enzymatic center, where no sensitive amino acid side chains are located.

[ 3 H] Proline and [ 3 H] uridine were injected into both eyes of the goldfish. 1 h before and after this injection 3 × 10 -6 м ouabain was administrated unilaterally to the retina. 8 h and 24 h after tracer injection the radioactivity in the retina, optic nerve and tectum was measured. It is suggested that the inhibition of the neuronal Na + -K + -ATPase inside the retina is responsible for the reduction of the labelled material transported into the optic nerve.

Euglena gracilis Klebs (strain Z) was grown heterotrophically in the dark for 6 days. Subsequently, the cells were grown photoautotrophically under white fluorescent light with varying amounts of 3-(3,4-dichlorophenyl)-1,1dimethyl urea (DCMU) (1.7 × 10 -9 -1.0 × 10 -5 mol/1) added to the nutrient medium. Under non-photosynthetic conditions (addition of 10 -6 -10 -5 mol DCMU/1), the cells still produced chloroplast structures and a considerable percentage of compounds typical for chloroplasts, such as galactolipids (40-50%), chlorophylls (40%), and polyunsaturated C 16 and C 18 fatty acids (16:2, 16:3, 16:4, 18:2, 18:3) (65%). The results indicate that under these conditions the above compounds are synthesized from carbon sources outside the chloroplasts.

Membrane-Bound Enzyme Complexes, Anacystis nidulans, Thylakoids, Benzoate Synthase The photosynthetic procaryote Anacystis nidulans converts L-phenylalanine and L-tyrosine into benzoic acid and p-hydroxybenzoic acid, respectively. Results obtained with thylakoid fractions support the hypothesis that the reaction sequence is catalyzed by thylakoid-bound enzyme complexes consisting of phenylalanine ammonia-lyase and benzoate synthase or tyrosine ammonia-lyase and p-hydroxybenzoate synthase, respectively. Both complexes do not accept phenylacetic acids as substrates, and cinnamic acids only at a small extent. These properties suggest a striking similarity to a benzoic acid-synthesizing enzyme system from higher plants which is situated at the thylakoid membrane of chloroplasts. The respective complexes of Dunaliella marina and Porphyridium sp. were included in this comparison.

The release of a part of the cell coat of Ehrlich ascites cells by chelation with ethylenediamine­ tetraacetic acid modifies significatively the uptake of various radioactive molecules. This phenomenon seems to be related to a change in the permeability of the cell membrane. The chemical analyses of the released material appear to indicate that glycoproteins are involved in that change and that their release modifies the structural and physiological characteristics of the cell membrane.

Fragmented sarcoplasmic reticulum (FSR) vesicles from rabbit muscle were suspended in 1.5 - 5% glycerol solutions and were pelleted onto aluminum foil disks in a modified centrifuge tube. Examination of these pellets in the electron microscope after drying for 2 - 2.5, 4 - 5.5, and 21 hours revealed a progression of changes. First, distances between individual, round vesicles decreases. Next, somewhat flattened vesicles establish limited areas of contact with adjacent vesicles. Finally, vesicle fusion occurs and extended areas of double bilayers are formed. A water loss-time interaction appears to be needed for the fusion process. A Hg-phenyl azoferritin com­ pound was used as a marker to identify intra- and extra-vesicular space in the fused samples. Quantitative measurements of birefringence during imbibition of pellet slices in a graded series of glycerol solutions indicates a steadily increasing amount of birefringence until 60 - 80% glycerol (η = 1.41 -1.43) is readied. The plateau seen in this part of the curve is again followed by steadily increasing birefringence at higher glycerol concentrations. This interruption in the birefringence curve is presumably due to a matching of the refractive indices of the glycerol solution and a lipid component in the membranes.

Membrane Fusion, Sarcoplasmic Reticulum, Ethylene Glycol, Dimethylsulfoxide Fragmented sarcoplasmic reticulum (FSR) vesicles from rabbit skeletal muscle were suspended in 5 - 10% ethylene glycol (EG) or in 5, 10, or 25% dimethylsulfoxide (DMSO) and were pelleted onto flat aluminum foil disks. No vesicle fusion occurs with either 5 or 10% EG treatment and 2-2½ hours drying. After 4-5 hours drying, 5% EG-treated vesicles have established more areas of close contact and individual vesicles have begun to flatten when compared with the 10% EG vesicles. Overnight drying results in formation of extended sheets of fused membranes called double bilayers in both the 5 and 10% EG samples. In the 5% EG-treated vesicles, formation of large spaces in the pellet compresses the adjacent bilayer pairs. Extensive vesicle fusion accompanies 4 - 5 hours drying of both 5 and 10% DMSO-treated samples. No fusion was observed in the 25% DMSO-treated sample after 4-5 hours drying and individual vesicles remained round as in the 2-2½ hour samples of all treatments. Overnight drying also causes extended sheets of bilayer pairs to form in the 5% DMSO-treated samples but, with 10 and 25% treated vesicles, destroys the double bilayers and only occasional dense regions of membrane whorls remain. Both EG and DMSO promote more rapid fusion of FSR vesicles than does glycerol but overnight drying after treatment with 10 or 25% DMSO destroys the fused membrane.

The 690 nm absorption change reflecting the turnover of the system-II-reaction center chlorophyll, Chl-a II (often referred to as P 680), has been investigated under different experimental conditions in spinach chloroplasts. A comparison was made with oxygen evolution and with absorption changes of Chl-a I measured at 703 nm, both indicating the number of electrons produced by system II. It was found: 1. The dependency on actinic flash intensity of the initial amplitudes of the measured 690 nm absorption change, ∆A 0 (Chl-an), markedly differs for normal and for Tris-washed chloroplasts, respectively. 2. The saturation curve of ∆A 0 (Chl-an) in Tris-washed chloroplasts is similar to that for the total amplitude of the 703 nm absorption change, ∆A 0 (Chl-a I ), in normal chloroplasts, and can be described by an exponential function. On the other hand, ∆A 0 (Chl-aII) in normal chloroplasts exhibits a more complex biphasic dependency and much higher flash intensities are required for saturation. 3. Under repetitive flash group excitation and in the presence of an A D R Y (= acceleration of the deactivation reactions of the water-splitting enzyme system Y)-reagent the initial amplitude of the 690 nm absorption change oscillates in the same characteristic pattern as the oxygen evolution. 4. The initial amplitude of the 690 nm absorption change, ∆A 0 (Chl-a II ), in Tris-washed chloroplasts becomes significantly smaller (more than 50%) by the addition of system-II-electron donors (benzidine, p-phenylendiamine, tetraphenylboron), whereas the total amplitude of the 703 nm absorption change, ∆A 0 (Chl-a I ) increases 3 -4-fold. In order to explain these results, the existance of a very fast reduction kinetics of Chl-a II + is postulated, which is not detectable by our measuring equipment. The half time of this reaction is ≦ 1 μs. Reaction centers with the very fast “undetected” Chl-a II + -reduction are photochemically transformed into slower one by double hit processes with a comparatively low quantum yield. Furthermore, it is inferred, that the dark recovery kinetics of Chl-a II is dependent on the charge accumulation state of the watersplitting enzyme system Y. This phenomenon is shown to explain also the oscillation pattern of delayed fluorescence. On the basis of the present results two alternative reaction schemes for the functional organization of the electron transport on the donor side of system II are discussed.

The chlorophyll fluorescence of isolated chloroplasts in the presence of phenazine methosulfate (PMS) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) can be quenched in a light-dependent reaction. This phenomenan has been studied and the following observations were made: 1. Quenching occurs under non-phosphorylating conditions and is stimulated by Mg 2+ ions. 2. Under the same conditions, a light-dependent, Mg 2+ stimulated transient decrease of absorp­ tion at 388 nm is observed which shows the spectral characteristics of PMS. 3. PMS is reversibly bound to chloroplasts. Under the experimental conditions used, binding amounts to as much as 0.5 mol PMS/mol chlorophyll. 4. Some uncouplers of photophosphorylation such as carbonylcyanide-m-chlorophenylhydrazon (CCCP) and atebrin analog abolish quenching, transient absorption change and binding of PMS. Others, such as methylamine, ammonia, gramicidin and nigericin do not. It is suggested that fluorescence quenching, transient absorption change and binding of PMS are causally related. The concept, postulated by others, that a high-energy state of the chloroplast membrane is involved in the fluorescence lowering is questioned.

Pharmacological studies on gastropod smooth muscle preparations have led us to the conclusion that the muscles in vitro behaves as if supplied by both excitatory and inhibitory cholinergic receptor sites.

It is suggested to use living cells (red blood cells, lymphocytes and leucocytes) as drug delivery systems for temporal and spatial drug administration in human therapeutics and diagnosis. The effectiveness of drug loaded cells is demonstrated for the drug methotrexate which is used in cancer treatment. Red blood cells are loaded with methotrexate using the dielectric breakdown technique. Dielectric breakdown leads to a transient increase of permeability of the cell membrane. Red blood cells loaded with tritium-labelled methotrexate were injected into mice and the activity level was measured in several organs as a function of time. It is shown that with this drug delivery system more than 50% of the drug (after 10 min) can be accumulated in the liver and that a high activity level can be sustained in this organ for more than 3 hours. On the other hand, administration of this drug by injecting solutions in the usual manner leads only to an 25% accumulation of methotrexate (after 10 min) in the liver. The drug is excreted completely after 1 to 2 hours. It is proposed to load red blood cells simultaneously with para-or ferromagnetic substances to obtain organ-specificity for any selected site of the body.

A dansyl (diaminonaphthalenesulfonyl)-derivative of cerebroside was prepared which could be effectively incorporated into the plasma membranes of tissue culture cells and erythrocytes. The cells which had assimilated the glycolipid fluoresced intensely and could be observed under a fluorescent microscope. Cells were initially labeled rather homogeneously over the whole surface. With longer incubation time organization of the fluorescent glycolipid took place and patches of the lipid in the membrane were formed. The redistribution and organization of the membrane lipid could be demonstrated most clearly when cells labeled with this fluorescent glycolipid were infected with myxoviruses. After infection of MDBK and BHK cells with fowl plague virus areas of dense fluorescence appeared at margines of neighboring cells. When BHK cells were infected with Newcastle disease virus fusion of the cells was accompanied by complete redistribution of the glycolipid. Erythrocytes could also easily incorporate dansyl cerebroside. Chicken erythrocytes which contain cytoplasmic and nuclear membranes incorporated the fluorescent glycolipid in both membranes.

Ultraweak Photoemission, Biological Systems, Cell Communication Measurements on untreated yeast cells, potato-and cucumber seedlings have confirmed the existence of photon emission from biological systems known as “ultraweak luminescence”. The intensity is of the order of 10 2 counts per second, and the spectral distribution shows a maximum near 550 nm. The dependence of the photon emission on certain chemicals has been investigated. Some chemicals, as for instance acetone, intensify the photon emission from cucumber seedlings without essential change of the spectral distribution. On the other hand, NaCl leads to a shift to the red region of the spectrum. The treatment of EAT-cells by 4-Hydroperoxycyclofosfamide involves a nonlinear enhancement of the intensity with increasing concentration. There are some indications that collective interactions of the system are more appropriate than simple chemiluminescence reactions.

Langmuir-Blodgett type polypeptide monolayers have been irradiated under conditions simulating the hazards of electron microscopic imaging. The damaging effect, i. e. the randomization of the α-helical layers, which occurs in the dose range between 50 and 200 e - /nm 2 , has been monitored by infrared dichroism.

The terpenes camphene, camphor, isopulegol, isoborneol, bomeol and the terpenoid methyl 3-isopropyl-pentanoate are identified from both species while n-nonanal was only found in F. rufa.

Rate constants of the alkaline hydrolysis reaction were correlated with neurotoxic findings of some organophosphates using data published in the literature. This correlation shows upper and lower limits of reactivity comparable to those found with biological activities of some other xenobiotics.

The methylmercury-induced dealkylation of the corrinoid coenzyme methylcobalamin, yielding aquocobalamin and dimethylmercury as products, was studied spectrophotometri-cally at 350 nm using water as a solvent. Rate data were determined for the pH 7-9 region and also at pH 3.37. Evidence is provided which shows that CH 3 Hg + serves as the species which accepts the methyl group and also that Hg 2+ is methylated more rapidly than CH 3 Hg + is.

The cis,cis-muconic acid derivative of pyrazon, which was formerly isolated from the medium of pyrazon-de-grading bacteria, was formed enzymatically by incubation of the catechol derivative of pyrazon with partially purified ortho pyrocatechase from pyrazon-degrading bacteria.

By treatment of 2-hydroxymuconic acid with a partially purified 4-oxalocrotonate decarboxylase 4-hydroxy-2-oxo-valerate could be obtained. Both forms of 4-hydroxy-2-oxo-valerate, the keto as well as the enol form could be isolated.

Three forms of the amino terminal half (variable region) of human pathological lambda light chains of immunoglobulins were identified antigenically. By study of all completely sequenced Bence Jones proteins hitherto analyzed and a greater number of proteins subjected to automated sequence determination as well as normal light chains three distinct isotypic basic sequences were identified. The basic sequences are shown to be associated with characteristic antigenic markers representing three V region genes encoding the variable half of lambda chains of immunoglobulins.

The Yolk Nucleus in Young Oocytes of Neomacheilus barbatulus (L.) (Teleostei, Cobitidae) Yolk Nucleus, Oocytes, Electron Microscopy, Noemacheilus barbatulus The yolk nucleus in the young oocytes of the freshwater teleost Noemacheilus barbatulus (L.) was investigated by electron microscopy. Yolk nuclei are only found in stage I. They consist of nucleolar substance, mitochondria and partly of dictyosomes. The nucleolar material leaves the nucleus through the pores of the nuclear membrane and gets into the juxtanuclear cytoplasm. It mainly consists of ribonucleic acid. The yolk nuclei of Noemacheilus doesn’t have any relations to the formation of yolk.

Rhodopsin, Insect Photoreceptor, Freeze Fracture Electron-microscopic examination of freeze-fractured fly retinae has revealed the presence of particles, 80 to 100 Å in diameter, on the photoreceptor membrane. Flies which were raised on a vitamin-A deficient diet show a substantial reduction in the density of such particles. The reduction in particle density is in agreement with the reduction in visual-pigment concentration as measured spectrophotometrically for these flies. These results suggest that the particles are identical with molecules of the visual pigment, rhodopsin.

Compound Eye, Light-Quantum Noise The highly specialized dorsal compound eye of male Simuliids is especially adapted to detect during flight the females which are very small animals (1×3 mm). At dawn, the eye functions close to the physical limit imposed by the quantum fluctuations of light. In bright illuminations, light-qauntum noise is not a limiting factor as can be shown by behavioural experiments.

Chick embryo fibroblasts infected by a temperature sensitive mutant of Rous sarcoma virus were enucleated with Cytochalasin B. The cytoplasts were still able to transform morphologically when shifted from nonpermissive to permissive temperature, and to revert to normal morphology when shifted from permissive to nonpermissive temperature. This indicates that the viral gene product responsible for transformation acts primarily on cytoplasmic or membrane components and not on the nucleus.