Identification and characterization of a novel D-amidase gene from Variovorax paradoxus and its expression in Escherichia coli.

The gene for the newly described D-amidase from Variovorax paradoxus (Krieg et al. 2002) was cloned and functionally expressed in Escherichia coli. Since native enzyme was available in minute amounts only, we determined the N-terminal sequence of the enzyme and utilized the Universal GenomeWalker Approach to make use of the common internal sequence of the amidase signature family. The high GC content of the gene made it necessary to employ an appropriate DNA polymerase in the amplification reactions. Thus, the sequence of the complete gene and the flanking regions was established. In independent experiments, the gene was then amplified from genomic DNA of V. paradoxus, expressed in E. coli, and characterized. The recombinant enzyme has a specific activity of 1.7 units/mg with racemic tert-leucine amide as substrate and is a homodimer of 49.6-kDa monomers.

Differences in the susceptibility of plant membrane lipids to peroxidation.

Peroxidation of three membrane lipid preparations from plants was initiated using Fe-EDTA and ascorbate and quantified as the production of aldehydes and loss of esterified fatty acids. Using liposomes prepared from commercial soybean asolecithin, the degree of peroxidation was shown to be dependent on: the free radical dose, which was varied by the ascorbate concentration; the presence of tocopherol in the liposome; the configuration, of the liposome, multilamellar or unilamellar; and time after initiation. There were dramatic interactions among these factors which led to the conclusion that in comparing the susceptibility of different membrane preparations it is essential to examine the kinetics of the peroxidation reactions. The composition of the liposome was a major determinant of the degree of peroxidation and of the type of degradative reactions initiated by the oxygen free radicals. A fresh polar lipid extract from Typha pollen had very similar fatty acid composition to the soybean asolecithin, but was more resistant to peroxidation as shown by less aldehyde production and increased retention of unsaturated fatty acids after treatment. Similarly, microsomal membranes from the crowns of non-acclimated and cold acclimated winter wheat (Triticum aestivum L.) seedlings had a much higher linolenic acid content than soybean asolecithin but was much more resistant to peroxidation. In the winter wheat microsomes, the loss of esterified fatty acids was not selective for the unsaturated fatty acids; consequently, even with 40% degradation, the degree of unsaturation in the membrane did not decrease. These different reaction mechanisms which occur in plant membranes may explain why measurements of fatty acid unsaturation fail to detect peroxidative reactions during processes such as senescence, aging and environmental stress.

Growth, ammonia accumulation and glutamine synthetase activity in alfalfa (Medicago sativa L.) shoots and cell cultures treated with phosphinothricin.

Phosphinothricin is a non-selective herbicide which inhibits glutamine synthetase (EC 6.3.1.2) activity causing an overaccumulation of ammonia in higher plants. Alfalfa (Medicago sativa L) shoot tissue and petiole-derived callus exposed to phosphinothricin show 50 and 70% reductions, respectively, in glutamine synthetase activity with a concomitant rise of 10 and 20 fold, respectively, in endogenous ammonia. The diffusibility of ammonia may limit the use of a detoxifying gene, phosphinothricin acetyltransferase, as a selectable marker for alfalfa transformation. However, the addition of up to 40 times the standard levels of ammonium nitrate to the culture media used in this study had no effect on callus growth, although glutamine synthetase activity was inhibited by 50% and endogenous ammonia increased 27 fold. Therefore, ammonia accumulation may not be the primary cause of cell death in alfalfa after exposure to phosphinothricin. It follows that diffusion of ammonia from cell to cell would not restrict the selection for phosphinothricin acetyltransferase transformed cells, thereby indicating that this enzyme could be used as a selectable marker in transformation experiments.

Polysomal polyadenylated RNA and albumin messenger RNA in Mastomys liver and in a chemically induced hepatocellular carcinoma.

A hepatocellular carcinoma line (H78) was established from a primary liver tumor induced in Mastomys natalensis by a single administration of dimethylnitrosamine. Six to 8 months after transplantation (passages 5 to 7), well-differentiated tumors, still containing glucogen-storing cells, were isolated and used for the preparation of RNA. Polysomal polyadenylated RNAs from Mastomys liver and from H78 tumor line were then compared by hybridization kinetics. Total kinetic complexities were 6.6 X 10(9) and 6.3 X 10(9) daltons for liver and tumor, respectively. Complexities of the high and middle abundant class were reduced in the hepatoma. Heterologous hybridization reactions revealed that, in terms of RNA mass, all or most of the polysomal polyadenylated RNA present in the liver was also present in the tumor and vice versa. However, shifts in the relative abundance of messenger RNA sequences were detected. In contrast to most other transplanted hepatomas, H78 has approximately the same content of albumin messenger RNA on its polysomes as has untreated liver.

Poly(A)- and oligo(A)-adjacent sequences in RNA from the large hnRNP complexes of rat liver.

The present report describes the isolation of hnRNP complexes with sedimentation coefficients greater than 80 S from rat liver nuclei without the use of ribonuclease inhibitors. The RNA moiety from these complexes is heterogeneous in size, with a mean sedimentation coefficient of 16 S when assayed with polyacrylamide-formamide gel electrophoresis. About 3% of this RNA binds to oligo(dT)-cellulose, the size of the bound fraction being somewhat smaller than the bulk of the hnRNP-RNA. This poly(A) RNA contains two adenylate tracts, one with about 30 and the second with about 200 adenylate residues. Reverse transcription of the poly(A)-containing RNA and hybridization of the cDNA with their respective templates shows two well-defined frequency populations, the first one separated from the second by almost 2.5 logs in the R0t curve. The same distribution was found, whether the hybrids were analysed with S1 nuclease or hydroxyapatite. Both separated frequency classes hybridize to total genomic DNA, the abundant one at C0t values typical for repetitive and unique sequences, the scarce one only at C0t values typical for unique sequences. The two populations are also able to hybridize with polysomal polyadenylated mRNA, the dilution of both frequencies being very similar in the cytoplasm.

Albumin messenger RNA after partial hepatectomy and sham operation.

Albumin mRNA was quantified in nuclear RNA and in polysomal and post-polysomal poly(A)-containing RNA from untreated, hepatectomized and laparotomized rat livers. A prominent reduction of albumin-specific RNA sequences was observed in all subcellular fractions 50 h after partial hepatectomy, compared to both untreated and sham-operated livers. Surprisingly, laparotomy itself also induced a dilution of albumin-specific sequences at earlier times after operation in nuclei and in post-polysomal supernatant, but not in polysomes.

Kinetic complexity of nuclear poly(A)-containing RNA in normal and regenerating rat liver.

Poly(A)-containing RNA isolated from liver nuclei of untreated rats and 3 h or 12 h after partial hepatectomy or sham operation was hybridized to the complementary DNAs (cDNAs). In the homologous reactions two major components could be seen. When compared to normal liver, the complexity of the least abundant class was lower in nuclei from livers 3 h after partial hepatectomy and was higher in those isolated 12 h after operation. The heterologous reactions revealed an increase of some abundant poly(A)-containing sequences and a loss or dilution of rare sequences 3 h after operation. The latter effect was not specific to the regeneration process but occurred after laparotomy as well. 12 h after partial hepatectomy, however, about 10% new poly(A)-containing sequences were detected, corresponding to about 5000 molecules of 4500 nucleotides length, which are unique to regenerating nuclei.

[Chemotherapy in solid transplantation tumors following synchronization (author's transl)]. / Chemotherapie bei soliden Transplantationstumoren nach Synchronisation.

In order to examine whether the effects due to cytostatics might be improved by synchronization, cytosin-arabinoside, hydroxyurea or Vincristin were administered to rats bearing solid transplantation tumors, at different intervals following synchronization. The three substances, interfering with the S-phase of the cells, show significantly improved therapeutic results with quickly proliferating Walker-carcinosarcoma after a synchronization by hydroxyurea. Cyclophosphamide on the other hand, being effective through several phases of the cell cycle, furnishes no evidence of any essential therapeutic result. Analogous therapeutic experimentation using slowly growing neurosarcomas yielded no better results after synchronization. The concept of synchronization treatment may be approved, therefore, with certain limitations: firstly, synchronization with hydroxyurea can be maintained for a short time only, and the cancerous cells pass throught the first phases of the cell cycle following synchronization for the most part together with normal cells; secondly, only tumors with an important growth fraction and only cytostatics that do not affect several phases of the cell cycle are appropriate for synchronization therapy.