Studies on tissue cultures of the genus Cinchona L. alkaloid production in cell suspension cultures

Initiation and culture of callus and cell suspensions of Cinchona ledgeriana and C. succirubra as well as the successful isolation and selection of a high-yielding alkaloid-forming strain derived from the leaf rachis of a C. succirubra plant are described. Results of feeding experiments with L-tryptophan using two different culture procedures are presented and discussed. Maximum alkaloid yields of up to 0.9% (based on dry weight) or 6.35 mg/l have been obtained.     

Indole alkaloids from cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga

From cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga grown under standard conditions, six monoterpenoid indole alkaloids have been isolated and identified. T. divaricata synthesized apparicine, catharanthine, coronaridine, conoflorine, tubotaiwine and vinervine, whereas T. iboga produced tubotaiwine and conoflorine. Both cultures are a reasonable source for conoflorine, which is expected to be a good candidate for studying the mechanism of Aspidosperma type alkaloid formation at the cell-free level.     

The oxidation-reduction kinetics of the reaction of cytochrome c1 with non-physiological redox agents

The kinetics of the oxidation-reduction reactions of cytochrome c₁ with ascorbate, ferricyanide, triphenanthrolinecobalt(III) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) have been examined using the stopped-flow technique. The reduction of ferricytochrome c₁ by ascorbic acid is investigated as a function of pH. It is shown that at neutral and alkaline pH the reduction of the protein is mainly performed by the doubly deprotonated form of ascorbate. From the ionic-strength-dependence studies of the reactions of cytochrome c₁ with ascorbate, ferricyanide and triphenanthrolinecobalt(III), it is demonstrated that the reaction rate is governed by electrostatic interactions. The second-order rate constants for the reaction of cytochrome c₁ with ascorbate, ferricyanide, TMPD and triphenanthrolinecobalt(III) are 1.4·10⁴, 3.2·10³, 3.8·10⁴ and 1.3·10⁸ M^?1·s^?1 (pH 7.0, I = 0, 10°C), respectively. Application of the Debye-Hückel theory to the the ionic-strength-dependence studies of these redox reactions of cytochrome c₁ yielded for ferrocytochrome c₁ and ferricytochrome c₁ a net charge of ?5 and ?4, respectively. The latter value is close to that of ?3 for the oxidized enzyme, calculated from the amino acid sequence of the protein. This implies that not a local charge on the surface of the protein, but the overall net charge of cytochrome c₁ governs the reaction rate with small redox molecules.     

Der Einfluss von CCC auf die Entwicklung des Roggenhalmes

Field trials on the effect of chlorocholinechloride (CCC) on rye plants of the cultivar Danae and of a selected population “WRS” proved that rye principally shows as reaction analogous to wheat. The CCC-induced decrease of stalk length is due to the reduction of elongation growth of the 4th internode. This shortening effect is mainly the result of decreased cell extension and, in the middle internode, additionally of inhibited cell division in longitudinal direction. The shape of internodes is changed under the influence of CCC. Walls of parenchyma cells of CCC-treated plants are thinner and those of sclerenchyma cells are thicker compared with cell walls of control plants.      

Light scattering,chlorophyll fluorescence and state of the adenylate system in illuminated spinach leaves

Scattering of green light and chlorophyll fluorescence by spinach leaves kept in a stream of air or nitrogen were compared with leaf adenylate levels during illumination with blue, red or far-red light. Energy charge and ATP-ADP ratios exhibited considerable variability in different leaves both in the dark and in the light. Variability is explained by different possible states of the reaction oxidizing triose phosphate or reducing 3-phosphoglycerate. Except when oxygen levels were low, there was an inverse relationship between light scattering and chlorophyll fluorescence during illumination with blue or red light. When CO₂ was added to a stream of CO₂-free air, chlorophyll fluorescence increased, sometimes after a transient decrease, and both light scattering and leaf $\text{ATP}\text{ADP}$ ratios decreased. Similar observations were made when air was replaced by nitrogen under blue or high-intensity red light. Under these conditions, over-reduction caused inhibition of electron transport and phosphorylation in chloroplasts. However, when air was replaced by nitrogen during illumination with low-intensity red light or far-red light, light scattering increased instead of decreasing. Under these light conditions, $\text{ATP}\text{ADP}$ ratios were maintained in the light. They decreased drastically only after darkening. Although $\text{ATP}\text{ADP}$ ratios responded faster than light scattering or the slow secondary decline of chlorophyll fluorescence due to illumination, it appeared that in the steady state, light scattering and chlorophyll fluorescence are useful indicators of the phosphorylation state of the leaf adenylate system at least under aerobic conditions, when chloroplast and extrachloroplast adenylate systems can effectively communicate.     

Replacement of natural polyamines by cadaverine and its aminopropyl derivatives in Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma cells were cultured in the presence of difluoromethyl ornithine (DFMO) and micromolar concentrations of cadaverine for several months. This treatment resulted in a complete disappearance of putrescine and spermidine and reduced spermine content to traces of its normal content. The natural polyamines were replaced by cadaverine (about 40% of total polyamines), N-(3-aminopropyl)cadaverine (about 50%) and N,N′-bis(3-aminopropyl)cadaverine (about 5%). In comparison with untreated cells or cells grown in the presence of DFMO and putrescine, the “cadaverine cells” grew definitely slower, their protein synthesis was depressed while DNA and RNA syntheses proceeded at near normal rate. In spite of the high intracellular concentrations of cadaverine and its aminopropyl derivatives, the tumor cells grown in the presence of DFMO and cadaverine, behaved exactly like cells severly depleted of putrescine and spermidine. Though exposed to DFMO, ornithine decarboxylase activity was almost 10 times higher than that in untreated cells. S-Adenosyl-L-methionine decarboxylase activity was likewise strikingly elevated, and these cells transported methylglyoxal strikingly elevated, and these cells transported methylglyoxal bis(guanylhydrazone) (MGBG) at a rate that was more than 5 times faster than that in untreated cells. Furthermore, these cells exhibited arginase activity, which was less than one fifth of that found in untreated cells.     

Partial characterization of a specific high affinity binding macromolecule for 24R,25 dihydroxyvitamin D3 in differentiating skeletal mesenchyme

Cytosol preparations and cells from 6-day old cultured differentiating chick limb-bud mesenchyme, which consist of a high proportion of chondrocytes, were shown to specifically bind 24R,25 dihydroxycholecalciferol. Nuclei from identical cultures also showed specific binding for 24R,25 dihydroxycholecalciferol. On the contrary, similar preparations of limb-bud mesenchyme cells (6-day old cultures) pretreated on day one by 5-bromodesoxyuridine which induced a fibroblast phenotypic expression, failed to show any specific binding for either 24R,25 or 1α,25 dihydroxycholecalciferol. Pronase treatment of the cytosol indicated that the receptor was protein-like in nature. The chromatographic properties of the protein-receptor on diethylaminoethyl cellulose and Sephadex G-100 columns were similar to those of the protein receptor found for 1α,25 dihydroxycholecalciferol. This report is the first demonstration that a cytosol protein receptor for 24R,25 dihydroxycholecalciferol exists in developing skeletal tissue. 24,25 dihydroxyvitamin D₃ but not any of the other metabolites was shown to induce DNA synthesis after 24 h by almost two-fold and protein synthesis after 5 h by 240%. These results suggest an important physiological role for 24R,25 dihydroxyvitamin D₃ in the development of skeletal tissue.     

Activation of human T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate: role of accessory cells and interaction with lectins and allogeneic cells

Stimulatory effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocytes have been investigated. TPA was found to stimulate highly purified T cells (obtained by a three-step isolation procedure involving plastic adherence, nylon wool passage and Ig-anti-Ig column passage) in the absence of accessory cells (stimulation index of 5 to 10), whereas phytohemaglutinin (PHA) and concanavalin A (Con A) did not. This response was, however, increased by the addition of autologous adherent cells. Addition of TPA, but not adherent cells, induced T-cell proliferation in response to the nonmitogenic lectin, wheat germ agglutinin (WGA), while both adherent cells and TPA restored T-cell proliferation to mitogenic lectins such as PHA and Con A. Furthermore, TPA greatly increased the mixed-lymphocyte response of purified T cells to otherwise nonstimulating allogeneic cells such as T lymphocytes or tumor cells from some patients with chronic lymphocytic leukemia. These results suggest that TPA can directly act on human T cells to render them reactive to a variety of stimuli.     

Induction and repair of DNA strand breaks in cultured human fibroblasts exposed to various phenols and dihydrodiols of benzo[a]pyrene

Cultured human fibroblasts from healthy donors were incubated for 30 min with nine different benzo[a]pyrene (BP) derivatives in the presence or absence of liver microsomes from 3-methylcholanthrene treated rats. The induction and repair of DNA strand breaks were analysed by alkaline unwinding and separation of double and single stranded DNA (SS-DNA) by hydroxylapatite chromatography immediately after the incubation or at various times after the treatment. In the absence of microsomes DNA stand breaks were detected in fibroblasts exposed to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP) and the three BP dihydrodiols (BP-4,5-, BP-7,8- or BP-9,10-dihydrodiol). After removal of the BP derivatives from the medium the DNA strand breaks disappeared within 24 h. alpha-Naphthoflavone (alpha-NF) caused a decrease in the induction of strand breaks by 1-, 3- and 9-OH-BP but did not affect the induction of strand breaks in cells exposed to BP-7,8-dihydrodiol. In the presence of microsomes DNA strand breaks were found after exposure to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP), as well as BP-7,8- and 9,10-dihydrodiol. In contrast BP-4,5-dihydrodiol did not induce strand breaks under these conditions. The induction of strand breaks by BP-7,8-dihydrodiol was enhanced in the presence of cytosine-1-beta-D-arabinofuranoside (AraC). In all cases the DNA strand breaks had disappeared 24 h after removal of the BP derivatives and microsomes except after treatment with BP-7,8-dihydrodiol.