The moss genes <Emphasis Type="Italic">PpSKI1</Emphasis> and <Emphasis Type="Italic">PpSKI2</Emphasis> encode nuclear SnRK1 interacting proteins with homologues in vascular plants

The yeast Snf1, animal AMPK, and plant SnRK1 protein kinases constitute a family of related proteins that have been proposed to serve as metabolic sensors of the eukaryotic cell. We have previously reported the characterization of two redundant SnRK1 encoding genes (PpSNF1a and PpSNF1b) in the moss Physcomitrella patens. Phenotypic analysis of the snf1a snf1b double knockout mutant suggested that SnRK1 is important for the plant’s ability to recognize and adapt to conditions of limited energy supply, and also suggested a possible role of SnRK1 in the control of plant development. We have now used a yeast two-hybrid system to screen for PpSnf1a interacting proteins. Two new moss genes were found, PpSKI1 and PpSKI2, which encode highly similar proteins with homologues in vascular plants. Fusions of the two encoded proteins to the green fluorescent protein localize to the nucleus. Knockout mutants for either gene have an excess of gametophores under low light conditions, and exhibit reduced gametophore stem lengths. Possible functions of the new proteins and their connection to the SnRK1 kinase are discussed.     

Sediment toxicity in the Kattegat and Skagerrak

Sediments were sampled from 62 sites in the Kattegat and Skagerrak, which are located between the Baltic and the North Sea in the Western Atlantic, during autumn 1989 and spring 1990. From each site 5 to 6 samples were taken wit ha box-corer. After mixing to composite samples on board, transport and storage (at 4 °C for 2 to 4 weeks), the samples were tested for toxicity to Daphnia magna and Nitocra spinipes. Immobility in Daphnia after exposure to 16 percent sediment (wet wt) in reconstituted standardized water (ISO, 1982) ranged from 0 to 88 percent after 24 h and from 3 to 95 percent after 48 h. For Nitocra the toxicity, determined as the 96-h LC50 (% wet wt) at 7 salinity, ranged from > > 32 percent (nontoxic) to 1.8 percent (most toxic). All exposures were made in duplicates and the effects obtained in the duplicates with the same sediment were correlated to each other. However, sediment toxicity to Daphnia and Nitocra was not. The test with Nitocra, which was made at several concentrations of sediment, was considered to give the most reliable picture of sediment toxicity in the Kattegat and Skagerrak. This ambient toxicity assessment identified three areas with toxic sediment, (1) the Göta älv estuary (outside the city of Göteborg) and its surroundings, (2) the Bay of Laholm in southern Kattegat, which is an area with periodic oxygen depletion and where repeated mussel kills have occurred during the last decade, and (3) an area in the open Skagerrak northwest of Skagen (the tip of the Jutland peninsula). Sediments, which had been stored at 4 °C, were tested again after 6 to 13 mos with the Nitocra test. Stored sediment toxicity was poorly correlated with fresh sediment toxicity. The average detoxification during storage was 5 times, but the range was 3 orders of magnitude, from 17 times more toxic to 73 times less toxic. The reasons for the observed areal and storage differences in sediment toxicity are so far not understood.     

Activation of human T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate: role of accessory cells and interaction with lectins and allogeneic cells

Stimulatory effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocytes have been investigated. TPA was found to stimulate highly purified T cells (obtained by a three-step isolation procedure involving plastic adherence, nylon wool passage and Ig-anti-Ig column passage) in the absence of accessory cells (stimulation index of 5 to 10), whereas phytohemaglutinin (PHA) and concanavalin A (Con A) did not. This response was, however, increased by the addition of autologous adherent cells. Addition of TPA, but not adherent cells, induced T-cell proliferation in response to the nonmitogenic lectin, wheat germ agglutinin (WGA), while both adherent cells and TPA restored T-cell proliferation to mitogenic lectins such as PHA and Con A. Furthermore, TPA greatly increased the mixed-lymphocyte response of purified T cells to otherwise nonstimulating allogeneic cells such as T lymphocytes or tumor cells from some patients with chronic lymphocytic leukemia. These results suggest that TPA can directly act on human T cells to render them reactive to a variety of stimuli.     

Microbe-plant competition, allelopathy and arctic plants

Michelsen et al. (1995) present results of an experiment in which aqueous leaf extracts of three arctic woody plant species were found to inhibit growth and nutrient acquisition of three graminoid species, and suggested that microbial nutrient immobilisation, rather than allelopathy, was responsible for the observed trends. In doing this they also question previous work proposing that the Arctic dwarf shrub Empetrum hermaphroditum is allelopathic. We suggest that their conclusions are not unequivocally supported by their data. Firstly we indicate that the approaches used for estimating microbial nutrient immobilisation are questionable. Secondly we indicate that most of the trends that they discussed are based on data in which the treatments and controls are not significantly different for the majority of cases. Finally we respond specifically to their criticisms of previous work on E. hermaphroditum. While the question of how arctic plants interact is an interesting one, we conclude that this question cannot be answered by their data. Received: 20 November 1995 /Accepted: 10 July 1996     

A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs

Fatty acids, which are essential nutritional components, are also involved in cardiovascular and metabolic diseases. Here we report a human cell surface receptor that we name free fatty acid receptor (FFAR), because it is specifically activated by medium to long-chain free fatty acids. The receptor belongs to the class of seven-transmembrane, G-protein coupled receptors (GPCRs) and also mediates responses to antidiabetic drugs of the thiazolidinedione type. It is expressed in skeletal muscle, heart, liver, and pancreatic beta-cells. Stimulation of FFAR increases the intracellular calcium concentration in cells expressing the receptor in a native (pancreatic beta-cell line) or in a recombinant form. In view of the nature of the activating substances, their physiological role in the body, and the tissue distribution of FFAR we suggest the term "nutrient sensing receptor" for receptors acting at the interface between dietary components and signalling molecules.     

Acinetobacter baumannii Repeatedly Evolves a Hypermutator Phenotype in Response to Tigecycline That Effectively Surveys Evolutionary Trajectories to Resistance

The evolution of hypermutators in response to antibiotic treatment in both clinical and laboratory settings provides a unique context for the study of adaptive evolution. With increased mutation rates, the number of hitchhiker mutations within an evolving hypermutator population is remarkably high and presents substantial challenges in determining which mutations are adaptive. Intriguingly however, hypermutators also provide an opportunity to explore deeply the accessible evolutionary trajectories that lead to increased organism fitness, in this case the evolution of antibiotic resistance to the clinically relevant antibiotic tigecycline by the hospital pathogen Acinetobacter baumannii. Using a continuous culture system, AB210M, a clinically derived strain of A. baumannii, was evolved to tigecycline resistance. Analysis of the adapted populations showed that nearly all the successful lineages became hypermutators via movement of a mobile element to inactivate mutS. In addition, metagenomic analysis of population samples revealed another 896 mutations that occurred at a frequency greater than 5% in the population, while 38 phenotypically distinct individual colonies harbored a total of 1712 mutations. These mutations were scattered throughout the genome and affected ~40% of the coding sequences. The most highly mutated gene was adeS, a known tigecycline-resistance gene; however, adeS was not solely responsible for the high level of TGC resistance. Sixteen other genes stood out as potentially relevant to increased resistance. The five most prominent candidate genes (adeS, rpsJ, rrf, msbA, and gna) consistently re-emerged in subsequent replicate population studies suggesting they are likely to play a role in adaptation to tigecycline. Interestingly, the repeated evolution of a hypermutator phenotype in response to antibiotic stress illustrates not only a highly adaptive strategy to resistance, but also a remarkably efficient survey of successful evolutionary trajectories.     

Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.     

Real-time monitoring of rolling-circle amplification using a modified molecular beacon design

We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2′-O-Me-RNA to prevent 3′ exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.     

Glucose-stimulated efflux of fura-2 in pancreatic beta-cells is prevented by probenecid

Fura-2 loaded pancreatic beta-cells, isolated from obese hyperglycemic mice, were studied with respect to cytoplasmic free Ca2+ concentration ([Ca2+]i), insulin release and efflux of indicator. In the absence of glucose there was a continuous efflux of fura-2, which was markedly increased by stimulation with a high concentration of the sugar. Probenecid both reduced basal efflux of fura-2 and prevented that promoted by glucose. There was no interference of the drug with glucose-induced either insulin release or rise in [Ca2+]i. When applying fura-2 in pancreatic beta-cells, the use of probenecid markedly improves the measurements of [Ca2+]i.