Rapid cultivation of stable aerobic phenol-degrading granules using acetate-fed granules as microbial seed

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

Stimulus change dis-habituates operant responding supported by water reinforcers

The present experiment examined whether habituation contributes to within-session decreases in operant responding for water reinforcers. The experiment asked if this responding can be dishabituated, a fundamental property of habituated behavior. During baseline, rats’ lever pressing was reinforced by water on a variable interval 15-s schedule. During experimental conditions, rats responded on the same schedule and a new stimulus was introduced for 5 min at 15, 30 or 45 min into the 60-min session. The new stimulus was extinction, continuous reinforcement or flashing lights in different conditions. Rate of responding primarily decreased within the session during baseline. Introducing a new stimulus sometimes suppressed (extinction, continuous reinforcement) and sometimes increased (flashing lights) responding while it was in effect. The new stimulus increased responding after it ended and before it was presented in the session. The results are incompatible with the idea that non-habituation satiety factors (e.g., cellular hydration and blood volume) contributed to within-session changes in responding. These satiety factors should increase with increases in consumption, decrease with decreases in consumption and remain constant with constant consumption of water. Nevertheless, all stimulus changes increased operant responding for water. These results support the idea that habituation contributes to within-session decreases in responding for water reinforcers.     

Ganglioside GD1a restores infectibility to mouse cells lacking functional receptors for polyomavirus

Recent investigations on the pathway of cell entry by polyomavirus (Py) and simian virus 40 (SV40) have defined specific gangliosides as functional receptors mediating virus binding and transport from the plasma membrane to the endoplasmic reticulum (B. Tsai et al., EMBO J. 22:4346-4355, 2003; Gilbert and Benjamin, in press). These studies were carried out with C6 rat glioma cells, a heterologous host chosen for its known deficiency in ganglioside biosynthesis. Here, a cell genetic approach was undertaken to identify components required for the early steps of infection using mouse cells as the natural host for Py. Receptor-negative (R-) mouse cells, screened based on resistance to Py infection, were shown to bind Py but failed to allow entry of the virus. R- cells were also found to be resistant to SV40. Infectibility was restored or enhanced by the addition of the same specific gangliosides found in earlier studies with C6 cells. In one R- line, overexpression of caveolin-1 also increased infectibility. These results support and extend findings on gangliosides in lipid rafts as functional receptors and mediators of internalization for Py and SV40.     

Complementarity in the supramolecular design of arenaviruses and retroviruses revealed by electron cryomicroscopy and image analysis

Arenaviruses are rodent-borne agents of diseases, including potentially lethal human hemorrhagic fevers. These enveloped viruses encapsidate a bisegmented ambisense single-stranded RNA genome that can be packaged in variable copy number. Electron cryomicroscopy and image analysis of New World Pichinde and Tacaribe arenaviruses and Old World lymphocytic choriomeningitis virus revealed pleomorphic enveloped particles ranging in diameter from approximately 400 to approximately 2,000 A. The surface spikes were spaced approximately 100 A apart and extended approximately 90 A from the maximum phospholipid headgroup density of the outer bilayer leaflet. Distinctive stalk and head regions extended radially approximately 30 and approximately 60 A from the outer bilayer leaflet, respectively. Two interior layers of density apposed to the inner leaflet of the viral lipid bilayer were assigned as protein Z and nucleoprotein (NP) molecules on the basis of their appearance, spacing, and projected volume. Analysis of en face views of virions lacking the GP-C spikes showed reflections consistent with paracrystalline packing of the NP molecules in a lattice with edges of approximately 57 and approximately 74 A. The structural proteins of retroviruses and arenaviruses assemble with similar radial density distributions, using common cellular components.     

Linkage disequilibrium in European elite maize germplasm investigated with SSRs

Information about the extent and genomic distribution of linkage disequilibrium (LD) is of fundamental importance for association mapping. The main objectives of this study were to (1) investigate genetic diversity within germplasm groups of elite European maize (Zea mays L.) inbred lines, (2) examine the population structure of elite European maize germplasm, and (3) determine the extent and genomic distribution of LD between pairs of simple sequence repeat (SSR) markers. We examined genetic diversity and LD in a cross section of European and US elite breeding material comprising 147 inbred lines genotyped with 100 SSR markers. For gene diversity within each group, significant (P<0.05) differences existed among the groups. The LD was significant (P<0.05) for 49% of the SSR marker pairs in the 80 flint lines and for 56% of the SSR marker pairs in the 57 dent lines. The ratio of linked to unlinked loci in LD was 1.1 for both germplasm groups. The high incidence of LD suggests that the extent of LD between SSR markers should allow the detection of marker-phenotype associations in a genome scan. However, our results also indicate that a high proportion of the observed LD is generated by forces, such as relatedness, population stratification, and genetic drift, which cause a high risk of detecting false positives in association mapping.     

Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae

Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged. In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strain's reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2. Replacement of the conserved His(174) with alanine within the C-terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro. Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth. Deletion mutants further revealed that the N-terminal region of MetAP2 (residues 2-57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth. Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N-terminal region to interfere with wild-type MetAP2 function.     

Measuring phase shifts in humans following a simulated time-zone transition: agreement between constant routine and purification methods

Twelve healthy participants were studied in an Isolation Unit. For the first 7 (control) days, subjects lived on UK time. Then the clock was advanced by 8 h, mimicking an eastward time-zone transition, and for days 8 to 12, participants lived on this new local time. Two constant routines (participants were not allowed to sleep, were restricted in movement, and ate regular, identical snacks) were undertaken, during the control days (days 3 to 4) and at the end of the experiment (days 11 to 12). Rectal temperature and activity were measured throughout, with activity used to correct the measured temperatures for the direct (masking) effects of the sleep-wake cycle. Phase changes of the temperature rhythm between the constant routines were assessed by cross-correlation and cosinor analysis. During days 8 to 10, the measured temperatures and those that had been corrected (purified) for masking were assessed by the same two methods, and the shifts were extrapolated to predict the values expected during the second constant routine. Individuals differed widely in the phase shifts of the temperature rhythm, but the correlations between the changes measured by constant routines and those estimated by the purification methods were high (r=0.771 to 0.903), and the differences between them were not significantly different from zero (p>0.24). Phase shifts of the measured (masked) temperature rhythm were poorer predictors of the shift obtained from the constant routines (r3+/-4.5 h). Limitations of the methods due to the variability of results are discussed, but we conclude that the mean phase shifts obtained from purified, but not raw, temperature data show acceptable agreement with those found using our version of the constant routine.