Functional Tn5393-Like Transposon in the R Plasmid pRAS2 from the Fish Pathogen Aeromonas salmonicida subspecies salmonicida Isolated in Norway

Tn5393c containing strA-strB was identified as part of R plasmid pRAS2 from the fish pathogen Aeromonas salmonicida subsp. salmonicida. This is the first time an intact and active transposon in the Tn5393 family has been reported in an ecological niche other than an agricultural habitat.     

Cyclic AMP-mediated suppression of normal and neoplastic B cell proliferation is associated with regulation of myc and Ha-ras protooncogenes

Cyclic AMP functions as a negative regulator of cell proliferation in a variety of cell systems. We show here that the proliferation of normal and neoplastic B cells can be inhibited by high intracellular levels of cAMP. Thus forskolin treatment of the neoplastic B precursor cell line Reh induced a rapid increase in the cAMP level, which was followed by an accumulation of cells in the G0/G1 phase of the cell cycle over a period of 2-3 days. Similar inhibition of Reh cell proliferation after 3 days was observed whether forskolin was present continuously or only during the first 5 hr. Both c-myc and c-Ha-ras protein levels were transiently down-regulated at 4 hr of forskolin treatment, suggesting that these protooncogenes play a role in the process leading to cAMP-mediated growth cessation. Northern-blot analysis showed that the steady-state levels of c-myc RNA rapidly declined in all phases of the cell cycle, to return to control levels within a time period of 24 hr. In contrast, the c-Ha-ras mRNA level was steadily maintained. Thus the expression of c-myc and c-Ha-ras protein was regulated at different metabolic levels. The reduced proliferative capacity of the B precursor cell line in the presence of forskolin was not linked to induced differentiation. This was judged from the lack of appearance of three different B cell differentiation markers; cytoplasmic immunoglobulin heavy chain and two antigens recognized by the monoclonal antibodies B1 (CD20) and HH1 (CD37). We also showed that forskolin partially inhibited the proliferation of normal B lymphocytes stimulated by anti-immunoglobulins (anti-mu) and B cell growth factor (BCGF). The burst of c-myc mRNA during activation of normal B cells was also reduced by forskolin.     

Quantifying food limitation of arthropod predators in the field

A method for quantifying food limitation of arthropod predators in the field is presented and applied to species of ground beetles (Carabidae) and sheet-web spiders (Linyphiidae) from a cereal field. Food limitation is expressed quantitatively as accumulated hunger (=starvation) by transforming 24-h food consumption at 20°C of animals newly collected in the field into days of starvation at 20°C. This is done by means of a reference curve relating 24-h food intake at 20°C to starvation periods (days) at 20°C. Such a reference curve was obtained for the carabid beetle Agonum dorsale in the laboratory. For other species the reference curve was modified with species-specific data. The procedure makes it possible to compare the feeding conditions of different species populations that are part of the same community. Hunger levels in the field for females of A.␣dorsale were equivalent to c. 15 days of starvation in early spring, c. 5 days in June, increasing to c. 10 days in late summer. Two ground beetles occurring during summer also showed increasing hunger levels from June to July, probably the result of a dry summer. Two spider species experienced a hunger level in the range of 4–8 days of starvation. Received: 15 August 1997 / Accepted: 22 January 1998     

Life table parameters affecting the population development of the woolly beech aphid, Phyllaphis fagi

A detailed study of the basic biological parameters affecting the population development of the woolly beech aphid, Phyllaphis fagi L. (Homoptera: Drepanosiphidae), was performed under field and laboratory conditions in order to obtain information about factors responsible for outbreak situations in forest nurseries. In the nurseries, the aphid eggs have found to hatch before budburst. The newly hatched nymphs were highly active in searching for feeding places, which resulted in higher nymph mortality in the first generation than in the following generations. Ten aphid generations were recorded in the nursery during one growing season. In the laboratory, development time, nymph mortality, reproduction, and adult lifetimes were assessed for P. fagi at four constant temperatures, i.e., 12, 15, 20, and 25 °C through four successive generations. Both temperature and aphid generation were found to affect life table parameters. The first generation, however, was less susceptible to changing temperatures than the following generations. Phyllaphis fagi reproductive effort was more dependent on temperature than on aphid generation. No significant difference was found in reproductive effort between apterous and alate females. The potential for outbreak situations therefore exists throughout the summer. The formation of winged morphs was not restricted to a few generations but rather continued for several months, with activity peaking in all years in mid-June. This had implications for migration to new seedlings in the nurseries. The highest r_m value was obtained at 20 °C. Nymphs born at higher temperatures were sometimes born deformed and unable to survive.     

Instability of standard PCR reference genes in adipose-derived stem cells during propagation,differentiation and hypoxic exposure

Background  

For the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained.     

Prediction of antibiotic activity and synthesis of new pentadecapeptides based on lactoferricins.

The antibacterial activity against Escherichia coli and Staphylococcus aureus has been studied for a number of modified pentadecapeptides based on lactoferricins of different origin. The peptides were classified by multivariate methods and quantitative structure-activity relationships (QSAR) were developed using theoretically derived variables for the amino acids. For the modified peptides based on bovine lactoferricin (LFB) a model was calculated and used for prediction of new peptides that were then tested for antibacterial activity in order to improve peptide activity and to check the validity of the model. Models were also calculated including lactoferricins of different origin. Theories of the mechanism of action of the peptides are briefly discussed.     

A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected.     

Structure-activity relationship study of anoplin.

Anoplin is a decapeptide amide, GLLKRIKTLL-NH2 derived from the venom sac of the solitary spider wasp, Anoplius samariensis. It is active against Gram-positive and Gram-negative bacteria and is not hemolytic towards human erythrocytes. The present paper reports a structure-activity study of anoplin based on 37 analogues including an Ala-scan, C- and N-truncations, and single and multiple residue substitutions with various amino acids. The analogues were tested for antibacterial activity against both S. aureus ATCC 25923 and E. coli ATCC 25922, and several potent antibacterial analogues were identified. The cytotoxicity of the analogues against human erythrocytes was assessed in a hemolytic activity assay. The antibacterial activity and selectivity of the analogues against S. aureus and E. coli varied considerably, depending on the hydrophobicity and position of the various substituted amino acids. In certain cases the selectivity for Gram-positive and Gram-negative bacteria was either reversed or altogether eliminated. In addition, it was generally found that antibacterial activity coincided with hemolytic activity.     

Biogeochemical and Molecular Signatures of Anaerobic Methane Oxidation in a Marine Sediment

Anaerobic methane oxidation was investigated in 6-m-long cores of marine sediment from Aarhus Bay, Denmark. Measured concentration profiles for methane and sulfate, as well as in situ rates determined with isotope tracers, indicated that there was a narrow zone of anaerobic methane oxidation about 150 cm below the sediment surface. Methane could account for 52% of the electron donor requirement for the peak sulfate reduction rate detected in the sulfate-methane transition zone. Molecular signatures of organisms present in the transition zone were detected by using selective PCR primers for sulfate-reducing bacteria and for Archaea. One primer pair amplified the dissimilatory sulfite reductase (DSR) gene of sulfate-reducing bacteria, whereas another primer (ANME) was designed to amplify archaeal sequences found in a recent study of sediments from the Eel River Basin, as these bacteria have been suggested to be anaerobic methane oxidizers (K. U. Hinrichs, J. M. Hayes, S. P. Sylva, P. G. Brewer, and E. F. DeLong, Nature 398:802–805, 1999). Amplification with the primer pairs produced more amplificate of both target genes with samples from the sulfate-methane transition zone than with samples from the surrounding sediment. Phylogenetic analysis of the DSR gene sequences retrieved from the transition zone revealed that they all belonged to a novel deeply branching lineage of diverse DSR gene sequences not related to any previously described DSR gene sequence. In contrast, DSR gene sequences found in the top sediment were related to environmental sequences from other estuarine sediments and to sequences of members of the genera Desulfonema, Desulfococcus, and Desulfosarcina. Phylogenetic analysis of 16S rRNA sequences obtained with the primers targeting the archaeal group of possible anaerobic methane oxidizers revealed two clusters of ANME sequences, both of which were affiliated with sequences from the Eel River Basin.     

Strains of the heterotrophic flagellate Bodo designis from different environments vary considerably with respect to salinity preference and SSU rRNA gene composition

The morpho species Bodo designis is widespread and abundant globally in highly contrasting terrestrial and aquatic ecosystems. Whether the forms of Bodo designis from contrasting environments are conspecific, i.e. largely genetically identical, or whether they merely share the external morphology is presently not known. We examined the ability of different strains of Bodo designis isolated from different environments at different geographical sites to survive and grow at a salinity range of 0.5-45%. The Bodo designis strains from marine, freshwater, and terrestrial environments showed a different ability to cope with altered physiological conditions. Most of the tested strains were only able to tolerate a small salinity range, whereas others were able to withstand all tested salinity levels. We further examined the phylogenetic relationship between the different strains by sequencing the small subunit (SSU) rRNA gene. The resulting phylogenetic analyses suggest a huge genetic variation within Bodo designis, and also imply that Dimastigella and Rhyncomonas are developed inside Bodo designis. If the biological species concept is used, the genetic differences as well as the physiological barriers between the different strains of Bodo designis, would suggest that they should be assigned to different species.