Ammonia Assimilation in Alnus glutinosa and Glycine max: SHORT-TERM STUDIES USING [N]AMMONIUM

The pattern of assimilation of NH₄⁺ by Alnus glutinosa, a N₂-fixing, nonleguminous angiosperm, was examined. Detached nodules, roots, and nodulated roots of intact plants were exposed to ¹³NH₄⁺ for up to 15 minutes. Glutamine was the most highly labeled compound at all times; the only other compound labeled significantly was glutamate. Similar results were obtained after incubating soybean (L. merr) nodules and roots with ¹³NH₄⁺. These observations and the results of pulse-labeling and inhibitor studies with nodules of Alnus were distinctly different from those predicted for the assimilation of NH₄⁺ via glutamine synthetase and glutamate synthase and suggest that glutamate dehydrogenase may play a major role in the assimilation of exogenously supplied NH₄⁺.     

Effects of lanthanum on calcium-dependent phenomena in human red cells.

Lanthanum (0.25 mM) does not penetrate into fresh or Mg2+-depleted cells, whereas it does into ATP-depleted or ATP + 2,3-diphosphoglycerate-depleted cells, into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca2+-efflux completely and inhibits (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg2+-depleted cells Ca2+-Ca2+ exchange is inhibited by lanthanum. Ca2+-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca2+-leak +/ S.D. amounts to 0.28 +/ 0.08 mumol/1 of cells per min, whereas in KC1 medium to 0.15 +/ 0.04 mumol/1 of cells per min at 2.5 mM [Ca2+]e and 0.25 mM [La3+]e pH 7.1. Lanthanum inhibits Ca2+-dependent rapid K+ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K+ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol. Lanthanum at 0.2--0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca2+-loaded cells, however, is blocked by lanthanum owing to Ca2+-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca2+ concentrations.     

Influence of the hormone analogue 13-NLE-molitin and of 1-methyl-3-isobutylxanthine on tone and cyclic 3',5'-AMP content of antral and duodenal muscles in the rabbit.

1. By the action of 1-methyl-3-isobutylxanthine (isobutyltheophylline, 2 - 3 × 10⁻⁴ M), the content of cyclic 3', 5'-AMP in the antral and duodenal muscles of the rabbit is increased by 72 % and 126 %, respectively; by 1.8 × 10⁻⁷ M 13-norleucine-motilin and 1.8 × 10⁻⁶ M acetylcholine it is not changed. 13-norleucine-motilin is an analogue of the recently discovered duodenal tissue hormone motilin and has identical effects. 1-methyl-3-isobutylxanthine has a more powerful inhibiting effect on phosphodiesterase than has theophylline.2. 3 × 10⁻⁴ M isobutyltheophylline reduces the tone of the duodenal muscle while simultaneously increasing the content of cyclic AMP and negates the tone-enhancing effect of nle-motilin on the duodenal muscle, while nle-motilin increases the muscle tone lowered by isobutyltheophylline.3. The basic tone of the antral muscle is not reduced by isobutyltheophylline. However, the contraction-promoting effect of nle-motilin after an increase in cyclic AMP due to isobutyltheophylline is significantly lower.4. It is assumed that the changes in the tone or in the response of the antral and duodenal muscles to nle-motilin observed after the administration of isobutyltheophylline, are due to the increase of cyclic AMP in the tissue.5. The antagonistic effects of cyclic AMP and motilin on the gastro-intestinal muscles might be of physiological importance for the regulation of the gastro-intestinal motor activity.     

Non-random intrachromosomal distribution of chromatid aberrations induced by X-rays,alkylating agents and ethanol in Vicia faba

A reconstructed karyotype of Vicia faba with all chromosomes individually distinguishable was treated with triethylene melamine (TEM), cytostasan (CYT) (a new benzimidazol nitrogen mustard), mitomycin C (MI), ethanol (EA) and X-rays. The distribution within chromosomes of induced chromatid abberations was non-random for all agents. The number of segments involved in aberration clustering corresponded to the number of sites representing constitutive heterochromatin, or the regions immediately adjacent to these, as evidenced by the position of Giemsa marker bands. Which of these potential regions of aberration clustering reacted with preferential involvement in aberrations was, in part at least, dependent upon the inducing agent used. It is argued that this may be due to differences in the base composition and/or molecular conformation of heterochromatic regions. Unexpectedly, the distribution pattern of chromatid aberrations induced by mitomycin C was found to be different from those after treatment with the alkylating agents TEM and cytostasan although mitomycin C is assumed to induce aberrations via alkylation. If mitomycin C-induced aberrations are indeed due to alkylation, this indicates that different alkylating agents do not necessarily result in identical patterns of abberation clustering. The other two alkylating agents and ethanol resulted in similar patterns of preferential distribution of abberations. X-Ray induced chromatid aberrations also showed a non-random intrachromosomal distribution, but the clustering was less pronounced than after treatment with the chemical agents.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Enzyme sensor-FIA-system for on-line monitoring of glucose, lactate and glutamine in animal cell cultures.

Enzyme sensors for glucose, lactate and glutamine were connected via flow-injection analysis (FIA) devices to two different bioprocesses. They were used for on-line process control of perfused bioreactor systems containing mammalian cell lines producing a monoclonal antibody and recombinant interleukin-2. The biosensor system gives direct access to important process data which can be used as control parameters for long term cell cultivation systems.     

Improving ancient DNA read mapping against modern reference genomes

ABSTRACT: BACKGROUND: Next-Generation Sequencing has revolutionized our approach to ancient DNA (aDNA) research, by providing complete genomic sequences of ancient individuals and extinct species. However, the recovery of genetic material from long-dead organisms is still complicated by a number of issues, including post-mortem DNA damage and high levels of environmental contamination. Together with error profiles specific to the type of sequencing platforms used, these specificities could limit our ability to map sequencing reads against modern reference genomes and therefore limit our ability to identify endogenous ancient reads, reducing the efficiency of shotgun sequencing aDNA. RESULTS: In this study, we compare different computational methods for improving the accuracy and sensitivity of aDNA sequence identification, based on shotgun sequencing reads recovered from Pleistocene horse extracts using Illumina GAIIx and Helicos Heliscope platforms. We show that the performance of the Burrows Wheeler Aligner (BWA), that has been developed for mapping of undamaged sequencing reads using platforms with low rates of indel-types of sequencing errors, can be employed at acceptable run-times by modifying default parameters in a platform-specific manner. We also examine if trimming likely damaged positions at read ends can increase the recovery of genuine aDNA fragments and if accurate identification of human contamination can be achieved using a strategy previously suggested based on best hit filtering. We show that combining our different mapping and filtering approaches can increase the number of high-quality endogenous hits recovered by up to 33%. CONCLUSIONS: We have shown that Illumina and Helicos sequences recovered from aDNA extracts could not be aligned to modern reference genomes with the same efficiency unless mapping parameters are optimized for the specific types of errors generated by these platforms and by post-mortem DNA damage. Our findings have important implications for future aDNA research, as we define mapping guidelines that improve our ability to identify genuine aDNA sequences, which in turn could improve the genotyping accuracy of ancient specimens. Our framework provides a significant improvement to the standard procedures used for characterizing ancient genomes, which is challenged by contamination and often low amounts of DNA material.     

Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.