A sensitive and inexpensive yeast bioassay for the mycotoxin zearalenone and other compounds with estrogenic activity

Zearalenone (ZON) is a nonsteroidal estrogenic mycotoxin produced by plant-pathogenic species of Fusarium. As a consequence of infection with Fusarium culmorum and Fusarium graminearum, ZON can be found in cereals and derived food products. Since ZON is suspected to be a cause of human disease, including premature puberty syndrome, as well as hyperestrogenism in farm animals, several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We developed a low-cost method for monitoring ZON contamination in grain based on a sensitive yeast bioassay. The indicator Saccharomyces cerevisiae strain YZRM7 is unable to grow unless an engineered pyrimidine biosynthetic gene is activated by the expressed human estrogen receptor in the presence of exogenous estrogenic substances. Deletion of the genes encoding ATP-binding cassette (ABC) transporters Pdr5p and Snq2p increases net ZON uptake synergistically. Less than 1 microg of ZON per liter of medium is sufficient to allow growth of the indicator strain. To prevent interference with pyrimidines potentially present in biological samples, we also disrupted the genes FUR1 and URK1, blocking the pyrimidine salvage pathway. The bioassay strain YZRM7 allows qualitative detection and quantification of total estrogenic activity in cereal extracts without requiring further cleanup steps. Its high sensitivity makes this assay suitable for low-cost monitoring of contamination of maize and small grain cereals with estrogenic Fusarium mycotxins.     

Beauvericin-induced channels in ventricular myocytes and liposomes

The antibiotic Beauvericin (BEA) was previously shown to express ionophoric properties under simple experimental systems. Its channel-forming activity was examined in inside-out patches of ventricular myocytes and synthetic membranes with the patch clamp and fluorescence imaging techniques. Current transitions to several open state levels were evident after wash-in. The BEA channel is cation-selective. Conductance and kinetics are presented for K(+) and Na(+) substates and main states. The pore was blocked by La(3+). In myocytes, the [K(+)](i) was reduced, while [Na(+)](i) and [Ca(2+)](i) increased, leading to cytolysis. These results indicate that BEA forms cation-selective channels in lipid membranes, which can affect the ionic homeostasis.     

Cutting edge: a crucial role for B7-CD28 in transmitting T help from APC to CTL

Although APC activation via CD40-CD40L signaling plays a critical role in enabling CD4(+) T cells to provide the "help" necessary for cross-priming of naive CTL, it is unclear how this makes the APC competent for priming. We have investigated the roles of B7-1/B7-2 and their receptors [corrected] CD28/CTLA-4 in cross-priming of CD4-dependent CTL in vivo. We find that both CD28 and B7-1/B7-2 are required for CD40-activated APC to cross-prime CTL, and that priming by CD40-activated APC was prevented by blockade of CD28. Conversely, augmenting CD28 signals with an agonistic Ab bypassed the requirement for CD4(+) T help or CD40 activation. Interestingly, blockade of the negative regulatory B7 receptor CTLA-4 failed to prime CTL in the absence of T help. These results support a model in which activation-induced up-regulation of B7 molecules on APC leads to increased CD28 signaling and a commitment to cross-priming of CD4-dependent CTL.     

Interstitial deletion of the short arm of chromosome 4. A phenotype distinct from the Wolf-Hirschhorn syndrome

In this paper we report a 3-month-old male newborn with marked hypotonia and an interstitial deletion of the short arm of chromosome 4 but with preservation of the 4p16 band (karyotype 46,XY,del(4)(pter----p15.3::p14----cen----qter). In contrast to patients with a pure 4p16 deletion this patient presented dysmorphic stigmata which were much more discrete than those found in the typical Wolf-Hirschhorn syndrome.     

Thiophilic interaction chromatography for supercoiled plasmid DNA purification

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.     

Performance of trees in forest canopies: explorations with a bottom-up functional-structural plant growth model

Here we present a functional-structural plant model that integrates the growth of metamers into a growing, three-dimensional tree structure, and study the effects of different constraints and strategies on tree performance in different canopies. The tree is a three-dimensional system of connected metamers, and growth is defined by the flush probability of metamers. Tree growth was simulated for different canopy light environments. The result suggest that: the constraints result in an exponential, logistic and decay phase; a mono-layered-leaf crown results from self-shading in a closed canopy; a strong apical control results in slender trees like tall stature species; the interaction between weak apical control and light response results in a crown architecture and performance known from short stature species in closed forest; correlated leaf traits explain interspecific differences in growth, survival and adult stature. The model successfully unravels the interaction effects of different constraints and strategies on tree growth in different canopy light environments.     

Numbers of myosatellite cells in white axial muscle of growing fish: Cyprinus carpio L. (Teleostei).

By means of transmission electron microscopy (TEM), the percentage of myosatellite cells was shown to decrease from about 6% in carp of 5 cm standard length (SL) to less than 1% in carp larger than 18 cm SL. The ratio between muscle nuclei and non muscle nuclei remained constant. These TEM data, combined with data on the amount of DNA per gram of muscle tissue and per nucleus, were used to calculate the numbers of myosatellite cells per gram of tissue (TEM-DNA method). Total numbers of myosatellite cells could be calculated from the TEM-DNA data and the calculated amounts of muscle tissue per fish. After a slight initial increase, the total number of myosatellite cells in the white axial muscle of a carp appears to be rather constant during the growth phase of the fish. But the myosatellite cells become more and more diluted over an increasing number of myonuclei with age. In addition, the reliability of two new light microscopic methods for the determination of numbers of myosatellite cells was examined. The percentages of myosatellite cells were determined by counting the numbers of total nuclei and of heterochromatic nuclei situated inside the muscle fibers' basal laminae which were stained using an antibody against laminin. These percentages were not significantly different from those determined with TEM. The yield of myosatellite cells per gram of muscle tissue, isolated with a previously developed dissociation method, showed a direct relation to the number of myosatellite cells calculated to be present in the tissue (TEM-DNA method), but at a 1% level. Both methods are alternative ways to determine numbers of myosatellite cells when it is impossible or difficult to use TEM (e.g., large sample sizes, combination with immunohistochemical methods).     

RNase-sensitive and RNase-insensitive protective components isolated fromListeria monocytogenes

Crude ribosomes were isolated fromListeria monocytogenes serotype 4b and separated into two fractions by molecular sieve chromatography. Chemical analysis indicated that fraction I contained cell envelope components while fraction II contained the ribosomes. Both fractions protected mice againstListeria, but only in combination with the adjuvant dimethyldioctadecylammonium bromide (DDA). RNase-treatment; but not proteinase K-treatment destroyed the protective properties of fraction II, and RNA purified from fraction II also induced protection. Protection induced by fraction I was not affected by either RNase- or proteinase K-treatment. Both subcutaneous and intraperitoneal, but not intravenous administration of fraction I, fraction 11, or purified RNA induced significant protection against intraperitoneal infection, the intraperitoneal route of administration being the most effective. All preparations induced high levels of protection 3 to 7 days after administration, but protection was already decreased after 14 days. Protection induced with RNA appeared to be biphasic, because it also protected mice 1 day, but not 2 days after administration. Protection induced with both fraction I and RNA was at least in part nonspecific, because both preparations also protected mice againstL. monocytogenes serotype 3,Streptococcus pneumoniae andPseudomonas aeruginosa. Results are discussed in relation to previous work with analogous preparations fromP. aeruginosa.     

Dissecting out the Complex Ca2+-Mediated Phenylephrine-Induced Contractions of Mouse Aortic Segments

L-type Ca²⁺ channel (VGCC) mediated Ca²⁺ influx in vascular smooth muscle cells (VSMC) contributes to the functional properties of large arteries in arterial stiffening and central blood pressure regulation. How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (V_m) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca²⁺ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca²⁺ release and the tonic contraction determined by Ca²⁺ influx. The latter component involves both Ca²⁺ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca²⁺ influx by small variations of the VSMC V_m causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca²⁺ release from non-contractile sarcoplasmic reticulum Ca²⁺ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca²⁺ release form contractile or non-contractile Ca²⁺ stores with concomitant Ca²⁺ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.