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.     

Transient changes in the pattern of food intake following a simulated time-zone transition to the east across eight time zones

Twelve healthy adults were studied, singly or in groups of up to four, in an Isolation Unit before (control days) and for 3 days after a simulated time-zone transition to the east across 8 time zones (the clock being changed from 15:00 to 23:00 h). Subjects were free to choose how to pass their waking hours (though naps were forbidden), and to eat what and when they wanted. A wide selection of food was provided, though the subjects had to prepare it. Subjects completed food intake questionnaire on waking and at 3 h intervals during the waking day. This questionnaire assessed the reasons for choosing not to eat a meal or, if a meal was eaten, the reasons for doing so, the type of meal chosen and the reasons for this choice, and subjective responses to the meal (hunger before, enjoyment during, and satiety afterwards). Subjects also recorded the incidence and degree of indigestion and jet lag at 3 h intervals after the time-zone transition. Following the time-zone transition, the subjects experienced significant amounts of jet lag and recorded a significant increase in the incidence of indigestion. They also showed significant changes in their pattern of food intake, but, whereas the patterns of food intake were no longer significantly different from control days by the third post-shift day, the symptoms of jet lag and indigestion were still present then. The distribution of daytime meals was significantly affected on the first post-shift day, with a redistribution of the times that the main, hot meals were eaten; these times indicated some influence of an unadjusted body clock. On this day also, the reasons for determining food intake continued to be dominated by hunger and appetite (hunger even increasing in the frequency with which it was cited), and the reason for not eating a meal, by a lack of hunger. On both control and post-shift days, there was a marked effect of meal type upon the responses to food intake, with cold food being rated least and large hot meals most when appetite before the meal, enjoyment during it, and satiety afterward were considered. However, evidence suggested that the degree to which larger hot meals were preferred to cold meals was significantly less marked after the time-zone transition. On control days, sleep was unbroken; whereas, after the time-zone transition, all subjects woke on at least one of the 3 nights studied. During the first post-shift night, about half of the subjects ate a meal, the reason given being that they were “hungry.” On those occasions when subjects woke but did not eat a meal, the reason cited was because they “could not be bothered” as frequently as because they were “not hungry.”. A simulated time-zone transition is associated with significant changes to the incidence of indigestion, pattern of food intake, and subjective responses to food. However, these changes are generally transient and are only weakly linked to the sensation of jet lag.     

Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1 proteins of two subtypes differ in their ability to support assembly

The influenza A virus hemagglutinin (HA) transmembrane domain boundary region and the cytoplasmic tail contain three cysteines (residues 555, 562, and 565 for the H3 HA subtype) that are highly conserved among the 16 HA subtypes and which are each modified by the covalent addition of palmitic acid. Previous analysis of the role of these conserved cysteine residues led to differing data, suggesting either no role for HA palmitoylation or an important role for HA palmitoylation. To reexamine the role of these residues in the influenza virus life cycle, a series of cysteine-to-serine mutations were introduced into the HA gene of influenza virus A/Udorn/72 (Ud) (H3N2) by using a highly efficient reverse genetics system. Mutant viruses containing HA-C562S and HA-C565S mutations had reduced growth and failed to form plaques in MDCK cells but formed wild-type-like plaques in an MDCK cell line expressing wild-type HA. In cell-cell fusion assays, nonpalmitoylated H3 HA, in both cDNA-transfected and virus-infected cells, was fully competent for HA-mediated membrane fusion. When the HA cytoplasmic tail cysteine mutants were examined for lipid raft association, using as the criterion Triton X-100 insolubility, loss of raft association did not show a direct correlation with a reduction in virus replication. However, mutant virus assembly was reduced in parallel with reduced virus replication. Additionally, a reassortant of strain A/WSN/33 (WSN), containing the Ud HA gene with mutations C555S, C562S, and C565S, produced virus that could form plaques on regular MDCK cells and had only moderately decreased replication, suggesting differences in the interactions between Ud and WSN HA and internal viral proteins. Analysis of M1 mutants containing substitutions in the six residues that differ between the Ud and WSN M1 proteins indicated that a constellation of residues are responsible for the difference between the M1 proteins in their ability to support virus assembly with nonpalmitoylated H3 HA.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

Joint Bayesian estimation of alignment and phylogeny

We describe a novel model and algorithm for simultaneously estimating multiple molecular sequence alignments and the phylogenetic trees that relate the sequences. Unlike current techniques that base phylogeny estimates on a single estimate of the alignment, we take alignment uncertainty into account by considering all possible alignments. Furthermore, because the alignment and phylogeny are constructed simultaneously, a guide tree is not needed. This sidesteps the problem in which alignments created by progressive alignment are biased toward the guide tree used to generate them. Joint estimation also allows us to model rate variation between sites when estimating the alignment and to use the evidence in shared insertion/deletions (indels) to group sister taxa in the phylogeny. Our indel model makes use of affine gap penalties and considers indels of multiple letters. We make the simplifying assumption that the indel process is identical on all branches. As a result, the probability of a gap is independent of branch length. We use a Markov chain Monte Carlo (MCMC) method to sample from the posterior of the joint model, estimating the most probable alignment and tree and their support simultaneously. We describe a new MCMC transition kernel that improves our algorithm's mixing efficiency, allowing the MCMC chains to converge even when started from arbitrary alignments. Our software implementation can estimate alignment uncertainty and we describe a method for summarizing this uncertainty in a single plot.