Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence

GDP-mannose pyrophosphorylase (GMPase, EC 2.7.7.22) catalyses the synthesis of GDP-D-mannose and represents the first committed step in the formation of all guanosin-containing sugar nucleotides found in plants which are precursors for cell wall biosynthesis and, probably more important, the synthesis of ascorbate. A full-length cDNA encoding GMPase from S. tuberosum was isolated. Transgenic potato plants were generated in which the GMPase cDNA was introduced in antisense orientation to the 35S promoter. Transformants with reduced GMPase activity were selected. Transgenic plants were indistinguishable from the wild-type when held under tissue culture conditions, however, a major change was seen 10 weeks after transfer into soil. Transgenic plants showed dark spots on leaf veins and stems with this phenotype developing from the bottom to the top of the plant. In case of the line with the strongest reduction, all aerial parts finally dried out after 3 months in soil, in contrast to the wild-type plants which did not start to senesce at this time. This coincides with a reduction of ascorbate contents in the transgenic plants, which is in agreement with the recently proposed pathway of ascorbate biosynthesis. Furthermore, leaf cell walls of the transgenic potato plants had mannose contents that were reduced to 30-50% of the wild-type levels, whereas the composition of tuber cell walls was unchanged. The glycosylation pattern of proteins was unaffected by GMPase inhibition, as studied by affinoblot analysis.     

Monitoring of chemotherapy-induced proteinuria using capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was investigated for its suitability to monitor proteinuria occurring during nephrotoxic drug therapy. Urine samples of tumor patients receiving chemotherapy consisting of carboplatin, etoposide, and ifosfamide were concentrated and desalted in microconcentrators and analyzed in two different alkaline CZE buffer systems. Reduction of electroosmotic flow (EOF) by the addition of putrescine increased the number of resolved protein peaks. Both CZE methods were linear between 2.5 and 50 μg/ml, exhibited satisfactory precision (relative standard deviation <10%) and were suitable for monitor the time course of proteinuria after chemotherapy administration. In contrast to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), CZE detected interindividual differences in protein patterns. Whereas these differences hampered a direct quantification of proteins in urine, they may contain information on the type or extent of kidney damage.     

Lead Distribution and Mobility in a Soil Embankment Used as a Bullet Stop at a Shooting Range

The distribution of lead in and below a soil embankment used as a stop butt for lead bullets at a sport shooting range for more than 30 years was investigated. A vertical profile, just behind the shooting target, was mapped by 54 soil samples characterized by contents of lead bullets, soil lead, and easily leachable lead as measured in a leaching test (L/S 2). At the target, the soil contained up to 40% metallic lead and 5 to 10% lead associated with the soil particles (<2?mm). The leaching test showed concentrations of dissolved lead in the range 5 to 20?mg/l. However, in the bottom of the stop butt (about 1?m lower than the target) soil lead was only slightly elevated, and no increase in lead was found below the stop butt in the original soil profile. In the lower part of the stop butt, pH was around 5, which is considered to favor lead migration, but in the soil samples with lead bullets present pH was between 6 and 7. The elevated pH values, probably caused by the corrosion of lead bullets, may have been a significant factor in limiting the migration of lead in the stop butt. The investigation showed that the lead in the stop butt did not affect the surroundings, but that the high lead content of the soil would require that this be treated as waste if the facility was abandoned.     

High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a K_i of approximately 4 μM. Uptake studies with [1,2-dimethyl-¹⁴C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a K_m value of 4 ± 1 μM and a maximum rate of transport (V_max) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance ¹³C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and γ-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83–90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.     

Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms

In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.     

Muricauda ruestringensis Has an Asymmetric Cell Cycle

Muricauda ruestringensis B1 is a Gram ‐negative, marine bacterium and a member of the Flavobacteriaceae family. It is characterized by long appendages, which appear at different stages of growth. At the outer end of these appendages there is a bulbous structure. Investigating the cell morphology of strain B1 during batch growth revealed a high diversity of cell types and sizes. Apart from small rod‐shaped cells and rods with appendages, there were large rods and spherical cells of different sizes as well as spherical cells which had fimbriae. To be able to study the cell cycle events, it was essential to monitor the population dynamics of the involved individuals. For this purpose, fluorochromising techniques, multi‐parametric flow cytometry, image analysis and fluorescence microscopy were used. It was demonstrated that all cell types displayed a broad variation in DNA content; the precise number of chromosomes varied depending on the growth phase. The assortment was testified to hold 16S rDNA sequence identity. The cultures consisted of subpopulations whose density within a Percoll gradient varied considerably, ranging from 1.028 to 1.070. Consolidating the results of the morphological data, the chromosome content and the density of the subpopulations at different growth stages enabled us to construct an asymmetric cell cycle for the growth of strain B1 under the specific culture conditions of our experiments.     

Multiple Comparisons of Treatments with Stable Multivariate Tests in a Two‐Stage Adaptive Design,Including a Test for Non‐Inferiority

The application of stabilized multivariate tests is demonstrated in the analysis of a two‐stage adaptive clinical trial with three treatment arms. Due to the clinical problem, the multiple comparisons include tests of superiority as well as a test for non‐inferiority, where non‐inferiority is (because of missing absolute tolerance limits) expressed as linear contrast of the three treatments. Special emphasis is paid to the combination of the three sources of multiplicity – multiple endpoints, multiple treatments, and two stages of the adaptive design. Particularly, the adaptation after the first stage comprises a change of the a‐priori order of hypotheses.     

Infection-induced chromatin modifications facilitate translocation of herpes simplex virus capsids to the inner nuclear membrane

Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1.