Band 30, Heft 1-2

The preparation of phlebiarubrone (1) and polyporic acid (13) starting with 1,2-dimethoxy-4.5-methylenedioxybenzene (3) is described.

Treatment of human placenta microsomes with phospholipase A or D inhibits the 17β-hydroxysteroid dehydrogenase [17β-HSD] activity, parallel with the hydrolysis of membrane phospholipids. The 17β-HSD activity of phospholipase treated microsomes is reactivated by synthetic phospholipids. The distribution of 17β-HSD activity in subfractions of original microsomes and of phospholipase treated microsomes obtained by zonal centrifugation was studied. Solubilization of the microsomal 17β-HSD was achieved by phospholipase A treatment. Two 17β-HSD were solubilized from human placenta microsomes by phospholipase A treatment and were further purified by ammonium sulphate precipitation, gel filtration on BioGel A-0.5 m, DEAESephadex chromatography and by isoelectric focusing. The enzymes were purified 25.8 and 17.4 times. The isoelectric points and molecular weights of the two 17β-HSD were determined. Both enzymes are of a 17β-HSD type. One of the 17β-HSD, however, was sensitive to estradiol- 17β, the other to testosterone. The question of whether the two enzymes constitute a monomer and a dimer of the same 17β-HSD or are completely different enzymes, is discussed.

Detailed enzyme kinetic parameters of the reactions catalyzed by the two 17β-hydroxysteroid dehydrogenases (17β-HSD), which were solubilized from the microsomes of human placenta by treatment with phospholipase A, followed by enrichment and separation were determined. Both enzymes are strictly substrate specific. The most active substrate of one of the 17β-HSD (fraction A) is estradiol-17β, the other 17β-HSD (fraction B) is sensitive to testosterone. Both NAD and NADP can serve as hydrogen transferring coenzymes, the latter giving about one-third of the initial rate of the former. With respect to the influence of temperature, different buffers and pH values, Michaelis constants [K m ] with estradiol-17β and testosterone as substrates, the solubilized and separated microsomal 17β-HSD behave like those isolated from the cytoplasmic fraction. The two 17β-HSD, after solubilization from the microsomal fraction of human placenta, enrichment and separation from each other, show only a little activity for the transfer of hydrogen between C 17 of estradiol-17β and C 17 of androstenedione. On the other hand, intact microsomes and an integrated system prepared by recombination of the 17β-enzymes by preincubation in phosphate buffer are able to catalyse very actively the transfer of hydrogen between estradiol-17β and androstenedione. The effect of temperature and time on the recombination of the two enriched and separated microsomal enzyme activities and the determination of the pH-optimum of the hydrogen transfer reaction are described. Finally it is proposed that the hydrogen transfer between steroid hormones represents an aspect of the true reaction mechanism of steroid hormones: Steroid hormones function as hydrogen transferring coenzymes by forming part of a chain of hydrogen carriers.

5-Acetyl-4-methyl-1-(β-D-ribofuranosyl) -imidazole-5′-phosphate reacts with diphenylphospho chloridate forming the asymmetrical pyrophosphate ester. This in turn reacts with tri-n-butylammonium phosphate yielding 5-acetyl-4-methyl-imidazole-riboside-5′-diphosphate and with tri-rcbutylammonium pyrophosphate to give the nucleotide triphosphate. 5-Acetyl-4-methyl-imidazole-riboside-5′-pyrophosphate shows in the test with pyruvate kinase a reaction rate three times slower than that of ADP; but the same K m as that of ADP. The ATP analogue is only about 10% as effective as ATP itself in the test with hexokinase, 3-phosphoglycerate kinase and gluconate kinase. Adenylate kinase and NAD + kinase show no activity when ATP is replaced by the nucleotide-triphosphate-analogue. In presence of ATP the analogue strongly inhibits the reaction of adenylate kinase.

Complex formation of deoxyribonucleic acid and 2-amino-benzo [c] cinnoline is demonstrated by spectroscopic means. The small molecule though uncharged seems to intercalate between the base pairs of the deoxyribonucleic acid like the acridines. The complex constant, however, is smaller by about two orders of magnitude. The influence of salt concentration and temperature on the equilibrium is investigated. The involvement of charge-transfer contribution is suggested.

The interaction of Cu 2+ with isonicotinic acid hydrazide (INH) and some of its derivatives has been investigated by means of electron spin resonance, nuclear magnetic resonance, and optical absorption studies. It could be shown that an interaction exists between the metal ions and the hydrazine side chain in addition to the very weak interaction with the ring nitrogen. The results obtained indicate the formation of a charge transfer complex.

NADP + reduction in isolated chloroplasts of spinach by photosystem I at the expense of various artificial donor systems is not inhibited by the plastoquinone antagonist dibromothymoquinone. The coupled ATP formation in such photoreductions is attributed to an artificial energy conserving site, i. e. a proton liberation during oxidation of the donor at the inner surface of the thylakoid membrane. Some donor systems for photosystem I are stimulated by uncouplers whereas others are not. The stimulation shows no correlation to the efficiency of the coupled photophosphorylation. Instead a correlation of the stimulation by uncouplers to the presence of an acidic OH-group in the donor molecule is seen. The uncoupler effect is therefore not explained by a release of electron transport control by the high energy state but rather by a pH-dependent distribution of the donor compound across the membrane. This is supported by the properties of donor systems in sonicated chloroplast particles with external oxidation sites of photosystem I.

The oxygen uptake which is observed when Rhodospirillum rubrum chromatophores are illuminated under air and in the presence of reduced 2,6-dichlorophenolindophenol (DCIP), 2,3,5,6-tetra-methyl-p-phenylenediamine (diaminodurene, DAD) or N,N′-tetramethyl-p-phenylenediamine (TMDP) depends on the electron-donor concentration according to the equation of Michaelis-Menten. The apparent K m for the donor is lowered by the electron-transfer inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) which causes therefore a stimulation of the rate of the reaction at non-saturating concentrations of the donors. In contrast, the ATP formation which takes place simultaneously to oxygen uptake does not show an enzyme-like dependence on donor concentration. Moreover it is inhibited by HQNO to a variable extent, depending on the particular donor present and on its concentration. Therefore it appears that the HQNO-sensitive phosphorylation is coupled to a cyclic flow which coexists and competes with the non-cyclic flow from donor to oxygen. In the presence of HQNO, substrates and uncouplers of ATP formation accelerate somewhat the rate of the oxygen uptake supported by reduced DCIP and DAD. Thus part of the HQNO-resistant phosphorylation seems to be associated with the non-cyclic flow from those two donors to oxygen. The lack of stimulation by phosphorylation or by uncoupling of the TMPD-supported oxygen uptake does not permit a conclusion as to whether this reaction is coupled to ATP forma­ tion or not. Another part of the HQNO-resistant ATP formation is independent of the presence of oxygen and appears to be associated to cyclic flows which bypass the HQNO site. This type of phosphorylation is most important in the presence of TMPD.

Formation of Nitrite from Hydroxylamine in the presence of illuminated chloroplast lamellae is inhibited by superoxide dismutase but not by catalase, indicating that the superoxide free radical ion and not H 2 O 2 is responsible for the oxidation of hydroxylamine. Decarboxylation of α-keto acids on the other hand is strongly inhibited by catalase but only slightly by superoxide dismutase. Light-dependent hydroxylamine oxidation and decarboxylation of α-keto acids can be used, therefor, as specific and sensitive probes for the determination of either the superoxide free radical ion or hydrogen peroxide, respectively. Photosynthetic oxygen reduction in the presence of ferredoxin, (monitored by the above method) yields both H 2 O 2 and O 2 ·- . The addition of an oxygen reducing factor (ORF, solubilized by heat - treatment of washed chloroplast lamellae) instead of ferredoxin, however, stimulates only the production of H 2 O 2 , while O 2 ·- - formation is not observed. The cooperation of ferredoxin and ORF during photosynthetic oxygen reduction by chloroplast lamellae apparently produces H 2 O 2 not only by dismutation of O 2 ·- , but also by a separate mechanism involving ORF.

Ethylene Biosynthesis, Chloroplasts, Superoxide Free Radical Isolated chloroplast lamellae from spinach produce ethylene in the dark from methylmercapto-propanal (MMP) or from 2-keto-4-methyl-mercaptobutyrate (KMB) only in the presence of both NADPH and ferredoxin. Anthraquinone-2-sulfonic acid can substitute for ferredoxin. Catalase, superoxide dismutase, ethanol and ascorbate are inhibitors of NADPH-dependent ethylene forma­ tion. Isolated NADP-ferredoxin reductase in the presence of NADPH, ferredoxin and an oxygen reducing factor (ORF, isolated by heat-treatment of chloroplast lamellae) catalyzes ethylene formation from the above substrates in the dark without chloroplast lamellae. From the results it is concluded that chloroplast lamellae in the dark can reduce oxygen monovalently at the expense of NADPH, with the production of the OH-radical as the reactive species responsible for ethylene formation from MMP of KMB.

Short pulses of red light induce in etiolated barley seedlings an enhanced synthesis of plastidic benzoquinones and vitamin K 1 , which can be reverted by subsequent irradiation with short pulses of far-red. As compared to the dark there is more formation of plastoquinone-9 than α-tocopherol. The enhanced formation of vitamin K 1 is coupled with a concomitant decrease in the level of the second naphthoquinone vitamin “K”. The data show that active phytochrome Pfr , also in its ground state, induces the light triggered lipoquinone synthesis which is connected to thylakoid formation. The red light induction of enhanced plastoquinone-9 synthesis cannot be fully reverted by subsequent far-red and seems to be a very fast phytochrome response.

Short time red pulses, given 6 times for 5 min within 36 h, induce in etiolated barley seedlings an enhanced synthesis of the main chloroplast carotenoids β-carotene, violaxanthine, lutein and neoxanthine. The level of antheraxanthine and zeaxanthine decreases by red light treatment. These red light effects are reverted by subsequent short time far-red pulses. The results show that the white light induced change in the accumulation rate of individual carotenoids is initiated and regulated by active phytochrome P fr . In the case of neoxanthin and zeaxanthin the red light effects cannot be fully reverted by far-red; this points to very fast phytochrome reaction.

The content of adenosine-3′,5′-cyclic monophosphate (cAMP) in leaves of Impatiens sultani Hook, was measured by a protein-binding assay. A preceding administration of the natural cyto­ kinin zeatin causes a diminution of the cAMP level. Administration of gibberellin (GA 3 ) does not bring about any variations. There is no indication that cAMP is acting as a secondary messenger of either phytohormone. Theophylline does not increase the content of cAMP found in the leaves.

Experiments concerning tumor transformation of plant cells or protoplasts need complete knowledge of the effects of media components. Here inhibiting effects of cellulase Onozuka, glucose, sucrose and mannitol (up to 0.3 M ) , of tissue culture media, and of the antibiotic aureo-mycin on the tumor initiation process in Kalanchoe leaves are reported. Also the influence of these substances on the growth of the tumor inducing Agrobacterium tumefaciens is studied. The results lead to the design of conditions which should be favourable to in vitro transformation.

It is shown that if the membrane potential of cells of Valonia utricularis was increased rapidly by applying ~500 μs current pulses, dielectric breakdown of the membrane occured when the potential reached a value of ~0.85 V. The breakdown phenomenon observed was not associated with global damage to the cell or its membrane. The process could be repeated after a short resealing time (~ 10s), many times on a single cell. The rapidity of the breakdown process (~1 μs) rules out the possibility that dielectric breakdown occurs by a mechanism similar to that of punch-through, which involves changes in ionic profiles.

Twenty students having a comparable typing skill and speed (about 150 strokes per min) performed series of typewritings with contents well familiar to them. These writings were examined for errors (error = deviation from a given program of stroke sequences). The errors could not be caused by ingnorance of writing or spelling of the words. Two categories of errors (“exchanges” = “Verwechslungen” and “omissions” = “Auslassungen”) were explained to result from special “faults” in the data processing controlling the typing process (Fig. 3). The occurrence of these types of errors dependend on interactions of the following factors: 1. Preferences of succession which (in general) related the first letter of an actual letter-combination with the second one (also) in contrary succession (i ⇄ e) or with other letters, not occurring in the combination (n → d ; n → g) (Tab. I ) . 2. Tendencies of the particular letters of an actual sequence of letters ( = word) to occur already before and/or after the right sequential position (sequential interval of this effect ≤ 4 strokes; temporat interval: about 1 sec; Figs 1, 2). 3. Correspondences in particular characteristics of special letters (vowel-vowel-exchange, etc.). 4. The notice to type as quickly as possible. Interactions of these factors resulted in erroneous “anticipations” and “postpositions” of letters.

It is shown that the knowledge of the E-vector direction of the linearly polarized light at any point of the sky alone is insufficient for the determination of the position of the sun. If the E-vector direction of a second point is not known the knowledge of at least one other parameter is necessary. This parameter might be the height of the sun over the horizon. With the knowledge of the height the infinite number of solutions for the sun’s position becomes reduced to two, or in special cases to one. These cases are derived.

Up to now, the microtopography of glucose and O 2 concentration in the intercapillary region of tumour tissue has been determined under the simplifying assumption that substrate consumption is constant up to the periphery of the envisaged cylindrical space around a capillary. The general diffusion field equation presented in this paper takes into account that substrate consumption decreases in the unsaturated region of cancer cell glycolysis and respiration so that - compared to former computations - the critical supply radii needed for maintained proliferation increase by a factor of almost 1.8. With the aid of its general solution and the discussed parameters, the laws governing substrate supply, cell kinetics and therapy mechanisms in the intercapillary region of intact and treated tumour tissue are presented and discussed. The essential results there are as follows: - Even in case of hyperglycemia it is glucose supply (and not the O 2 supply) which determines proliferation and proliferation rate. - Under the conditions of longtime-hyperglycemia (400 mg%), the tumour volume being accessible to the attack of cancerostatica or radiation, is almost three times as high as under standard conditions. - For glucose (and cancerostatica) the time constant for interactions between circulation and tumour tissue near the necrotic region is equal to or greater than 400 min. This is why stimulation of cancer cell glycolysis in the most therapy-resistant cancer cell portions (i. e., increased proliferation rate and tumour hyperacidifica­ tion) can only be achieved if hyperglycemia is maintained for 24 hours or more. - Therapeuti­ cally treated cancer tissue is characterized by the discussed changes in the diffusion field of glucose and O 2 as well as a drop in the interaction time constant for glucose.

The actions of cytoplasm (ultracentrifuged supernatant 105 000 x g) and of serum on the DNA synthesis (uptake of [ 3 H]TTP) by isolated nuclei of liver cells were studied. Cytoplasm from liver cells obtained at various intervals after partial hepatectomy revealed a variable effect only with isolated nuclei of liver cells from partially hepatectomized rats. Cytoplasm obtained from liver cells eight hours after p artial hepatectomy failed to show the usual inhibitory effect. Presum ably the inhibitory substances norm ally present in cytoplasm are inactivated within a certain time after partial hepatectomy. Serum of partially hepatectomized anim als enhanced the uptake of [ 3 H]TTP by nuclei of normal liver cells and those from partially hepatectom ized animals. The stim ulating effect was present only in the serum obtained between four and twelve hours after partial hepatectomy. It remains unclear whether the effects described are growth factors specific for the liver.

Following the intraventricular injection of 5-hydroxytryptamine into the lateral ventricles of conscious mice, the concentration of brain lactate rose immediately but then decreased significantly compared with the saline injected controls. The concentration of brain glucose increased 20 min after the injection of 5-hydroxytryptamine. These effects of 5-hydroxytryptamine on lactate and glucose were qualitatively similar to those found following the administration of methysergide. After the parenteral adm inistration of 5-hydroxytryptophan, the lactate levels showed a biphasic change but brain glucose was significantly decreased for up to 90 min following the injection. p -Chlorophenylalanine potentiated the effect of 5-hydroxytryptamine by further increasing glucose and decreasing lactate levels. After histamine had been injected into the ventricles, the concentrations of bound glycogen and lactate were decreased whereas free glycogen and glucose were raised. It is suggested th at 5-hydroxytryptamine, and possibly histamine, are involved with control of glycolysis in the mouse brain.

The influence of spermine on the potassium loss from yeast cells as a consequence of membrane-disturbing actions was investigated. Spermine interfered strongly in the action of membrane-active bactericides on yeast cells; this interference resembles the action of certain metal ions. Spermine provides a protection against the positively charged pararosaniline and accomplishes a strong potentiation of the action of the negatively charged sodium dodecyl sulphate.

The presence of exotoxin in Bacillus thuringiensis was demonstrated and its quantity in the cells determined. The concentration of exotoxin in the producing microorganism is approximately half the concentration of ATP. Exotoxin is produced at such a rate th at the cell excretes 1/5 to 1/4 of its exotoxin content into the medium per minute.

A theoretical calculation for quantitative analysis of photoreactions in membrane interfaces is described. The method utilizes the photovoltage arising by the illumination of a pigmented mem brane. Since the water-soluble substrate is only added to one side of the membrane an electrical double layer is produced. The corresponding photovoltage is quantified by the Gouy-Chapman theory. A possibility to investigate non-isotropic effects is discussed.

For autocatalysis coupled with diffusion the local periodic concentration profiles are influenced essentially by electrical transport and convection. When diffusion is negligible for Michaelis-Menten-kinetics equations are given to determine the kinetic parameters V max and K m from transport measurements.

The loosely bound proteins from hum an erythrocyte membranes induce an up to more than 10 3 -fold increase in the electrical conductivity of black lipid membranes made from oxidized cholesterol. The conductivity increase continues until the membrane breaks. Its magnitude insreases with in­ creasing protein concentration and salt concentration. The results are compared with earlier results obtained with the strongly bound proteins from erythrocyte membranes.