Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone     

The legumin gene family: a reconstructed Vicia faba legumin gene encoding a high-molecular-weight subunit is related to type B genes

Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamin/glutamic acid-rich region encoded by the C-terminal part of the chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342–350) and limited own data.     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Fast Cytoplasmic pH Regulation in Acid-Stressed Leaves

Induction of photosynthesis in leaves was prolonged, and steadystate photosynthesis was inhibited by very high CO₂ concentrationswhich cause cytoplasmic acidification. Prolonged exposure tohigh CO₂ relieved initially observed inhibition of photosynthesisat least partially. The sensitivity of carbon assimilation tohigh CO₂ was different in different plant species. Acidificationby CO₂ (or subsequent alkalization) was detected by measuringrapid CO₂-release from the tissue and by monitoring fluorescenceof pH-indicating dyes which had been fed to the leaves throughthe petiole. The results indicate that two different mechanismsoperate in leaves to achieve and maintain pH homeostasis. Rapidand efficient pH-adjustment is provided by proton/cation exchangeacross the tonoplast. Slower and less efficient regulation occursby formation or consumption of base. In the presence of highCO₂ concentrations, protons are pumped from the cytosol intoalready acidic vacuoles. In turn, vacuolar cations replace exportedprotons in the cytosol permitting bicarbonate accumulation andincreasing the pH of the acidified cytosol. Similarly effectiveand fast proton/cation exchange relieves acid-stress in thechloroplast stroma and permits photosynthesis to proceed withhigh quantum efficiency or high light-saturated rates in thepresence of CO₂ concentrations which would, in the absence offast cytoplasmic pH regulation, inhibit photosynthesis. By inference,proton/cation exchange must also occur across the mitochondrialboundary. After cytoplasmic pH adjustment in the presence ofhigh CO₂, removal of CO₂ results in transient cytoplasmic alkalizationand, subsequently, in the return of cytoplasmic pH values tolevels observed prior to acid-stress. In addition to fast pHregulation by rapid proton/cation exchange across biomembranes,slow base production (e.g. NH₃-formation) also contributes torelieving acid stress. Base produced in the presence of highCO₂ is rapidly consumed after removal of CO₂. Implications of the findings in regard to forest damage by potentiallyacidic air pollutants such as SO₂ are briefly discussed. (Received November 8, 1993; Accepted February 3, 1994)     

Light-stimulated proton transport into the vacuoles of leaf mesophyll cells does not require energization by the tonoplast pyrophosphatase

Photosynthetic characteristics of transgenic tobacco (Nicotiana tabacum L.) plants with a soluble pyrophosphatase in the cytosol of their leaf cells were compared to those of wild-type plants. Although the development of the transgenic plants was somewhat retarded compared to the wild type, as shown by stunted growth and delayed flowering, photosynthetic responses were comparable in transgenic and wild-type leaves of similar physiological age. In particular, light-dependent proton transport into the vacuoles of leaf mesophyll cells was not decreased in leaves of the transgenic plants, which did not contain pyrophosphate in the cytosol owing to the presence of a soluble pyrophosphase. This shows that light-stimulated proton pumping did not require the pumping activity of the tonoplast pyrophosphatase. Apparently, light-stimulated proton pumping can be based solely on the activity of the tonoplast ATPase.Abbreviation CDCF 5-(and 6-)arboxy-2,7-dichlorofluorescein This work was supported within the Sonderforschungsbereiche 176 and 251 of the University of Würzburg.     

Specificity and function of monoclonal antibodies directed against Ewing sarcoma cells

A selection of 16 monoclonal antibodies has been produced against a fresh Ewing's sarcoma (ES) tumor mixed with a permanent ES cell line. The majority of antibodies identify an 80-kDa molecule, which is not detected on healthy tissues except on certain cultured monocytes. One antibody recognize the CD2 ligand MIC2 and 2 antibodies (numbers 13 and 16) define a higher-molecular-mass antigen. Antibody 16 is also expressed on mesenchymal fibroblasts of bone marrow or fetal origin. Tumorspecific antigen expression is potentially linked to the chromosome 22 abnormality decribed in Ewing's sarcoma.     

Orotidine-5′-monophosphate decarboxylase fromPseudomonas aeruginosa PAO1: Cloning,overexpression, and enzyme characterization

Orotidine-5-monophosphate decarboxylase (OMPdecase) catalyzes the final step in pyrimidine biosynthesis, the conversion of orotidine-5-monophosphate (OMP) to uridine-5-monophosphate. ThepyrF gene, encoding OMPdecase, was isolated from a chromosomal library ofPseudomonas aeruginosa PAO1 by screening for complementation of anEscherichia coli and aP. aeruginosa pyrF mutant. The nucleotide sequence of a 2510-bp chromosomal DNA fragment, complementing both strains, was determined (EMBL accession number X65613). On this a 696-bp open reading frame capable of encoding the 24 kDa OMPdecase was identified. Despite a generally good correspondence to other OMPdecase sequences, theP. aeruginosa gene was unique in that it did not constitute part of an operon. ThepyrF gene was amplified by polymerase chain reaction, overexpressed in the pT7-7/E. coli BL21(DE3) system and purified to near electrophoretic homogeneity by anion exchange chromatography. Characterization of the purified enzyme revealed the following data, aK _m value for OMP of 9.91 M and an isoelectric point of 6.65. No major decrease in enzyme activity was observed in a pH range between 7.8 and 10.2. Gel electrophoresis under nondenaturing conditions suggested that the native form of OMPdecase is the dimer.     

Resynchronization of the Circadian Corticosterone Rhythm After a Light/Dark Shift in Juvenile and Adult Mice

Most of the extensive literature concerning the resynchronization of circadian rhythms after a Zeitgeber shift is devoted to the dependence of resynchronization on the mode of the shift and the strength of the Zeitgeber, as well as on the circadian function investigated. Ontogenetic influences have rarely been investigated. Therefore, we studied the resynchronization of several circadian rhythms in juvenile and adult female laboratory mice. We present here the results concerning the corticosterone rhythm. The daily rhythms were determined as transverse profiles (2-h intervals) before as well as 3, 7, and 14 days after an 8-h phase delay of the light/dark cycle produced by a single prolongation of dark time. The corticosterone concentration in serum was determined radioimmunologically. In the control animals the daily patterns were bimodal, with main maxima at the end of the light time and secondary ones just after lights on. Ontogenetic differences were small. In adult mice the amplitude was slightly increased due to an increase in the maximum values, and the time of highest hormone concentrations was slightly phase advanced. In juvenile mice, a distinct daily pattern with a phase position in relation to the light/dark cycle corresponding to that of control animals was present on the 3rd day after the Zeitgeber shift. The daily mean as well as the minimum and maximum values increased initially and reached the values of control animals during the second week. In adult animals, a pronounced daily rhythm with the normal phase position was present only at the 7th postshift day. The amplitude, daily mean, and maximum values were decreased, and the minimum values were increased. The initial values were not reached even after 2 weeks. The results show that resynchronization was faster in juvenile mice compared with adult mice. As a possible cause for the observed age-related differences, a not yet stabilized phase-coupling between various circadian rhythms is supposed.     

The first decade of oligotrophication of Lake Constance

In Lake Constance, after several decades of cutrophication, a decrease in phosphorus loading over the last decade has lead to a partial recovery from eutrophication. Here we analyse the shift in the taxonomic composition of phytoplankton during the first decade of oligotrophication in Lake Constance. During the 1980s, spring total P concentrations decreased from ca. 130 to less than 50 ·l^–1. This decrease was reflected by an approximately proportional decrease in summer phytoplankton biomass while spring phytoplankton biomass seemed unresponsive. Major taxonomic changes occured during both growth seasons. In spring, the proportion of diatoms, green algae and Chrysophyta increased while the proportion of Cryptophyta decreased. The summer trend was very different: the relative importance of diatoms decreased and Cryptophyta and Chrysophyta increased, while Chlorophyta reached their peak around 1985. These trends are also analysed at the genus level. Comparison with taxonomic trends during the eutrophication period shows the expected reversals in most cases. Comparison with other lakes shows general similarities, with the notable exception that Planktothrix rubescens has never been important in Lake Constance. The increase of diatoms during spring is attributed to their improved competitive performance with increasing Si:P ratios. Their decrease during summer is explained by the increasing silicate removal from the epilimnion by increasing spring populations.     

Electron Flow to the Intersystem Chain from Stromal Components and Cyclic Electron Flow in Maize Chloroplasts, as Detected in Intact Leaves by Monitoring Redox Change of P700 and Chlorophyll Fluorescence

The functional pool size of electrons in the intersystem chainof the chloroplasts of maize was estimated to be about 25 perP700 by the redox change in P700 with single- and multiple-turnoverlights under far-red light in intact leaves. This is about twicethe pool size observed in C₃ plants. Furthermore, the stromalpool size of electrons that can be donated to P700⁺ after actinicillumination was larger in maize leaves than in leaves of C₃plants, giving a maximum value of 225 electrons per P700. Maizeleaves showed an increase in the yield of modulated Chl fluorescenceafter turning off of actinic light, which confirms the donationof electrons in the dark to the intersystem chain from the stromaldonors that accumulated during actinic illumination. We proposethat the mesophyll chloroplasts are responsible for a high levelof electron-donating activity to the intersystem chain fromstromal donors such as triose phosphates and malate with NADPHas an intermediate. The level of P700⁺ under strong far-redlight was decreased after actinic illumination, suggesting theoperation of an actinic light-triggered cyclic electron flowin chloroplasts of the bundle sheath cells. (Received August 14, 1992; Accepted October 13, 1992)