Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Mikromorphologie und Orientierungsmuster epicuticularer Wachs-Kristalloide: Ein neues systematisches Merkmal bei Monokotylen

Based on SEM examinations of some 1600 species of monocotyledons out of 61 families the characteristics of their epicuticular waxes are described in this preliminary paper and illustrated by 20 SEM micrographs. Four basic types of waxes are recognized. Two of them occur throughout the monocotyledonous and dicotyledonous orders of angiosperms; two others are of high systematic significance and restricted to particular orders of the monocotyledons: The “Convallaria-Type” is characterized by fine wax platelets with parallel orientation, forming a pattern around the stomata reminiscent of electromagnetic field lines. This type is restricted to the three liliiflorous ordersAsparagales, Liliales, andBurmanniales. The “Strelitzia-Type” is characterized by massive compound wax rodlets; it is restricted to the superordersAreciflorae, Commeliniflorae, Zingiberiflorae, plus the two liliiflorous ordersBromeliales andVelloziales. The data are compared with the classifications ofDahlgren & Clifford (1982); they tentatively indicate the exclusion ofBromeliales, Velloziales, andTyphales from theLiliiflorae.     

Productivity and temperature biology of two snowbed bryophytes

Summary Chlorophyll distribution within the carpets, CO₂ gas exchange under controlled conditions, and heat resistance of the snowbed bryophyte Anthelia juratzkana (Limpr.) Trev. were investigated. Also the gas-exchange parameters of the co-occurring Polytrichum sexangulare Floercke were assessed. Only the uppermost 4 mm layer of Anthelia carpets contains sufficient pigments for photosynthesis. At light saturation and optimal temperatures (6–11°C) the maximum rates of CO₂ uptake are 0.7 mg CO₂ g^-1dw h^-1 in Anthelia and 1.5 mg CO₂ g^-1dw h^-1 in Polytrichum. Gas exchange reaches light saturation at about 300 E m^-2s^-1 in both species. At +2°C the light compensation point is reached at ca. 10E m^-2s^-1 and increases significantly with increasing temperature. The lower temperature compensation point is reached at-4°C in Anthelia and does not drop much below-5°C in Polytrichum. Anthelia cannot sustain net photosynthesis beyond 30°C and Polytrichum not beyond 32°C. Nine month storage under dark, cold and wet conditions does not affect the photosynthetic capability of Anthelia. As a response, however, the net photosynthesis rate is depressed due to an increase of the respiration rates. Polytrichum sexangulare did not tolerate the storage so well. The heat resistance limit of Anthelia is low (39°C). There is evidence that the distribution of the two bryophytes within snowbed communities is determined by their capability to make use of low light intensities and their low temperature demand for optimal photosynthetic rates. Being resistant to long lasting cold, wet, and dark conditions, Anthelia is particularly adapted to grow in the border zone along permanent snowpatches. Polytrichum is more productive and is therefore capable of competing successfully at sites which are less extreme and therefore accessible for higher plants.     

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.