Immunomagnetic separation as a final purification step of liver endothelial cells

Summary We describe a fast and reproducible method that can be used as a final step in obtaining pure populations of liver endothelial cells. This method employs endothelial cell specific lectin covalently bound to magnetic polystyrene beads (Dynabeads). Evonymus europaeus agglutinin (EEA)-coated Dynabeads were used to purify monkey liver endothelium from Percoll gradient separated nonparenchymal cells. EEA-coated beads were also successfully used to purify monkey aortic endothelial cells. The endothelial cells grew to confluence as a cobblestonelike monolayer, expressed Factor VIII related antigen, and incorporated acetylated-low density lipoprotein. The magnetic beads seemed not to modify the normal properties of the isolated endothelium, thus facilitating their use in experimental studies. This immunomagnetic separation technique may be applicable for purification of endothelial cells from a wide variety of tissue sources.     

Spatial population genetic structure in Trillium grandiflorum: the roles of dispersal, mating, history, and selection

The roles of the various potential ecological and evolutionary causes of spatial population genetic structure (SPGS) cannot in general be inferred from the extant structure alone. However, a stage-specific analysis can provide clues as to the causes of SPGS. We conducted a stage-specific SPGS analysis of a mapped population of about 2000 Trillium grandiflorum (Liliaceae), a long-lived perennial herb. We compared SPGS for juvenile (J), nonreproductive (NR), and reproductive (R) stages. Fisher's exact test showed that genotypes had Hardy-Weinberg frequencies at all loci and stage classes. Allele frequencies did not differ between stages. Bootstrapped 99% confidence intervals (99%CI) indicate that F-statistic values are indistinguishable from zero, (except for a slightly negative FIT for the R stage). Spatial autocorrelation was used to calculate f the average kinship coefficient between individuals within distance intervals. Null hypothesis 99%CIs for f were constructed by repeatedly randomizing genotypic locations. Significant positive fine-scale genetic structure was detected in the R and NR stages, but not in the J stage. This structure was most pronounced in the R stage, and declined by about half in each remaining stage: near-neighbor f = 0.122, 0.065, 0.027, for R, NR, and J, respectively. For R and NR, the near-neighbor f lies outside the null hypothesis 99%CI, indicating kinship at approximately the level of half-sibs and first cousins, respectively. We also simulated the expected SPGS of juveniles post dispersal, based on measured R-stage SPGS, the mating system, and measured pollen and seed dispersal properties. This provides a null hypothesis expectation (as a 99%CI) for the J-stage correlogram, against which to test the likelihood that post-dispersal events have influenced J-stage SPGS. The actual J correlogram lies within the null hypothesis 99%CI for the shortest distance interval and nearly all other distance intervals indicating that the observed low recruitment, random mating and seed dispersal patterns are sufficient to account for the disappearance of SPSG between the R and the J stages. The observed increase in SPGS between J and R stages has two potential explanations: history and local selection. The observed low total allelic diversity is consistent with a past bottleneck: a possible historical explanation. Only a longitudinal stage-specific study of SPGS structure can distinguish between historical events and local selection as causes of increased structure with increasing life history stage.     

Particulate formate oxidase from Nitrobacter agilis

1. The nitrite oxidase particles obtained by sonic oscillation of Nitrobacter agilis cells also possessed appreciable formate oxidase activity, ranging from about 25 to 50% of the nitrite oxidase activity depending upon the N. agilis strain. Both activities distributed themselves in the same pattern and proportions during differential centrifugation, and resided solely in the pellet resulting from high-speed centrifugation.

2. Difference spectra of formate-reduced particles or intact cells demonstrated the presence of cytochromes of the c- and a-types like those of the NO₂⁻-reduced material. Under anaerobic conditions NO₃⁻ or fumarate acted as an alternate electron acceptor in place of O₂ in formate oxidation. Under aerobic conditions increasing NO₃⁻ concentrations resulted in (a) an increased role of NO₃⁻ as a terminal electron acceptor compared to O₂, (b) a greater total enzymatic transfer of electrons from formate than if O₂ were the sole electron acceptor, and (c) a partial inhibition of O₂ uptake suggestive of a competition for electrons by the two acceptors. The formate oxidase system failed to catalyze consistently the transfer of electrons to either added mammalian cytochrome c or Fe(CN)₆³⁻. The marked sensitivity of the system to certain inhibitors implicated cytochrome oxidase as an integral part of the formate oxidase. The system was also inhibited significantly by a variety of chelating agents, indicating a metal component in the formate dehydrogenase or early portion of the electron transfer sequence.

3. The stoichiometry of the formate oxidase system was shown to approach the theoretical value of 2 moles of CO₂ evolved per mole of O₂ or per 2 moles of formate consumed.

4. To a limited extent, phosphorylation occurred concomittantly with the oxidation of formate in the presence of the cell-free particulate system.     

Widespread occurrence of spliceosomal introns in the rDNA genes of ascomycetes

Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.     

The evolution of California's wild radish has resulted in the extinction of its progenitors

If two previously isolated taxa mutually assimilate through hybridization and subsequent biparental introgression, and if their introgressed descendants have the same or higher fitness than their parents, then gene flow should result in the local extinction of parental taxa via replacement by hybrid derivatives. These dramatic events may occur rapidly, even in a few generations. Given the speed at which such extinction by hybridization may occur, it may be difficult to identify that the process has occurred. Thus, documented instances of extinction by hybridization are rare, and especially so for cases in which both parents are replaced by the hybrid lineage. Here we report morphological and allozyme evidence for the local extinction of two Raphanus species in California via replacement by their hybrid-derived descendants. The results from a greenhouse experiment demonstrate that California wild radishes have a specific combination of traits from their progenitors, and comparison of our results to that of an earlier report indicate that pure parental types are no longer present in the wild. Our results also show the hybrid-derived lineage has transgressive fruit weight compared to its parents. Allozyme analysis demonstrates that California wild radishes are derived from hybridization between the putative parental species. However, that analysis also demonstrates that California wild radish has now become an evolutionary entity separate from both of its parents. We suggest that the aggressive colonizing behavior of the hybrid-derived lineage probably results from a novel combination of parental traits, rather than genetic variability of the population per se.     

Flowering asynchrony and mating system effects on reproductive assurance and mutualism persistence in fragmented fig-fig wasp populations

? Premise of the study: Plants and animals may experience reproductive Allee effects in fragmented populations, and obligate pollination mutualisms may be especially sensitive to extinction risk via this density-dependent process. In this study we examine how a shift from within-crown reproductive synchrony to asynchrony influences reproductive assurance through contributions to selfing and outcrossing in small, spatially isolated populations of Ficus. ? Methods: The research focuses on the monoecious fig F. petiolaris and consists of phenological analyses and genetic assessments of selfing and outcrossing for populations located in Baja California's Sonoran Desert. ? Key results: Phenological censuses of eight populations revealed within-crown asynchrony in 44% of reproducing trees, with 16% having sufficient overlap of male and female flowering phases to permit selfing via the cycling of pollinating fig wasps within natal trees. In mating system analyses of two of these populations, however, multilocus outcrossing rates (t(m)) were indistinguishable from 1. This result, combined with low levels of inbreeding, indicates selfing to be absent or at best a minor contributor to reproductive assurance. ? Conclusions: The results indicate that the fitness benefits of within-crown asynchrony lie not with selfing, as commonly asserted, but with increased opportunities for outcross pollen transmission and receipt, changing our understanding of the mechanisms by which reproduction is facilitated and extinction risk minimized in naturally fragmented Ficus populations. Given the role of fig fruit as a keystone food resource in many tropical environments, trait variation leading to reproductive assurance in figs, such as within-crown asynchrony, has broader ecosystem-level implications.     

Decreased pre-existing Ad5 capsid and Ad35 neutralizing antibodies increase HIV-1 infection risk in the Step trial independent of vaccination

Background

The Step trial raised the possibility that uncircumcised men with pre-existing Ad5 neutralizing antibodies carried an increased risk of HIV infection after vaccination. Thus, understanding Ad seropositivity in humans is important to the development of an AIDS vaccine. Here, we analyze the impact of different Ad5-specific neutralizing antibodies on immune function and clinical outcome.

Methods and Findings

Ad seropositivity in the Step trial volunteers was analyzed using chimeric rAd5/35 vectors to characterize their specificity for Ad5 fiber and non-fiber external (capsid) proteins. Immune responses and HIV seropositivity were correlated with the specificity of Ad5-neutralizing antibodies. Neutralizing antibodies induced by the vaccine in Ad5 seronegative subjects were directed preferentially to Ad5 capsid proteins, although some fiber-neutralizing antibodies could be detected. Pre-vaccination Ad5 serostatus did not affect the capsid-directed response after three vaccinations. In contrast, anti-fiber antibody titers were significantly higher in volunteers who were Ad5 seropositive prior to vaccination. Those Ad5 seropositive subjects who generated anti-capsid responses showed a marked reduction in vaccine-induced CD8 responses. Unexpectedly, anti-vector immunity differed qualitatively in Ad5 seropositive participants who became HIV-1 infected compared to uninfected case controls; Ad5 seropositive participants who later acquired HIV had lower neutralizing antibodies to capsid. Moreover, Ad35 seropositivity was decreased in HIV-infected subjects compared with uninfected case controls, while seroprevalence for other serotypes including Ad14, Ad28 and Ad41 was similar in both groups.

Conclusions

Together, these findings suggest that the case subjects were less immunologically responsive prior to infection. Subjects infected during the Step trial had qualitative differences in immunity that increased their risk of HIV-1 infection independent of vaccination.