Abnormal expression of surface immunoglobulin isotypes on antigen-binding B lymphocytes from mice tolerant to trinitrophenyl determinants

Temporary B-cell tolerance to the trinitrophenyl (TNP) hapten can be produced in BDF1 mice by intraperitoneal injection of trinitrobenzene sulfonic acid (TNBS). Antigen-binding cells (ABC) specific to TNP, measured as TNP donkey erythrocyte rosettes, are found in tolerant mice as well as in immune mice. We have studied the surface immunoglobulin isotype profile of these TNP-binding lymphocytes (TNP-ABC) in four groups of animals: nonimmune, immune, tolerant, and tolerant-challenged. Immune mice received intravenous TNP sheep erythrocytes (TNP-SRC), whereas tolerant-challenged mice received TNP-SRC and TNBS on Day 0. TNP-ABC from mice immunized with TNP-SRC exhibit increased expression of surface IgG and decreased expression of surface IgD, compared to the ABC from nonimmune mice. Tolerant mice have a higher proportion of ABC with surface IgG, and a lower proportion with surface IgD, than nonimmune mice. Tolerant-challenged mice have a lower proportion of ABC with surface IgG, and a higher proportion with surface IgD, than immune mice. Thus, B-cell tolerance in this model entails an attenuation of the surface immunoglobulin isotype switch (loss of IgD and gain of IgG) on ABC seen in the normal immune response. For most TNP-ABC, tolerogen exposure prevents the switch in surface isotypes normally induced by exposure to TNP antigen; i.e., the tolerance lesion precedes the surface isotype switch. However, a minority of the TNP-ABC appear to switch surface isotypes in response to the tolerogen itself.     

Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis

Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the alpha3beta1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with alpha3beta1, maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu(149) and Glu(213)). Furthermore, the substitution of 11 amino acids (residues 195-205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151-CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin-tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151-alpha3beta1 interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151-alpha3beta1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the "tetraspanin web" can be controlled at various levels by different regions of the protein.     

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell-cycle synchronized in G0/G1 using serum starvation (serum-starved) was examined. Oocyte-karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum-starved group (79 vs. 56% respectively; P < 0.05). Development to the 4-cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum-starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum-starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum-starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty-five nuclear transfer embryos reconstructed using serum-starved cells were transferred to 15 temporarily mated recipients for 3-4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4-cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred.     

Resources and pollinators contribute to population sex-ratio bias and pollen limitation in Fragaria virginiana (Rosaceae)

Populations of gynodioecious species vary in the ratio of female versus hermaphroditic individuals they contain, and many exhibit higher frequencies of females under poor resource conditions. One important factor limiting female frequencies within populations is predicted to be pollen limitation of seed production, caused by either low abundance of pollen donors or insufficient pollen transfer. However, empirical studies measuring variation in pollen limitation with population sex ratios or resource gradients in gynodioecious plants are inconsistent. Part of this inconsistency may be that pollen limitation and its causes are context-dependent. Another possibility is that sex-specific daily flower production and/or sex-biased visitation are more relevant to the likelihood of pollen limitation than sex ratio based on counting individual plants. In this study, we examined context-dependent pollen limitation in gynodioecious/subdioecious Fragaria virginiana . We specifically examined the potential for resource availability to influence sex-specific daily flower production, sex-biased pollinator visitation, and their relationships with pollen limitation in experimental populations that contained either high or low frequencies of female plants. High resource availability reduced apparent female frequency by increasing daily flower production by hermaphrodites relative to females. This is important because pollinators increasingly discriminated against female flowers as floral sex ratios became more female-biased. Contrary to expectation, females in high-female populations were not consistently more pollen limited than those in low-female populations. The level of pollen limitation of females was better explained by sex–biased pollinator foraging and visitation frequency than by the plant sex ratio or floral sex ratio. Thus, negative frequency dependence of female pollen limitation was evident only considering sex ratio bias mediated by pollinator visitation.     

Genetic constraints on floral evolution: a review and evaluation of patterns

The characteristics of flowers influence most aspects of angiosperm reproduction, including the agents of pollination and patterns of mating. Thus, a clear view of the forces that mediate floral phenotypic evolution is central to understanding angiosperm diversity. Here, we inform on the capacity for floral phenotype to respond to selection by reviewing published data on heritabilities and genetic correlations for several classes of floral traits (primary sexual, attraction, mating system) in hermaphroditic plants. We find significant heritability for all floral traits but also variation among them, as well as a tendency for heritability to vary with mating system, but not life history. We additionally test predictions stemming from life history theory (eg, negative covariation between male-female traits and flower size-flower number), and ideas concerning the extent and pattern of genetic integration between flowers and leaves, and between the sexes of dioecious and gynodioecious species. We find mixed evidence for life history tradeoffs. We find strong support for floral integration and its relation with floral morphology (actinomorphy vs zygomorphy) and for a decoupling of floral and vegetative traits, but no evidence that modular integration varies with floral morphology. Lastly, we find mixed evidence for a relationship between the level of sexual dimorphism in attraction traits and the between-sex correlation in gender dimorphic plants.     

Unique Ion Signature Mass Spectrometry, a Deterministic Method to Assign Peptide Identity

The growing use of selected reaction monitoring (SRM) mass spectrometry in proteomic analyses led us to investigate how to identify peptides by SRM using only a minimal number of fragment ions. By using a computational model of the SRM work flow we computed the potential interferences from other peptides in a given proteome. From these results, we selected the deterministic SRM addresses that contained sufficient information to confer peptide and protein identity that we termed unique ion signatures (UIS). We computationally showed that UIS comprised of only two transitions are diagnostic for >99% of Escherichia coli proteins and >96% of human proteins that possess a sequence-unique peptide. We demonstrated an example of experimental use of UIS using a modified SRM methodology to profile the E. coli tricarboxylic acid cycle from a single injection of cell lysate. In addition, we showed the potential of UIS to form the first functionally orthogonal approach to validate peptide assignments obtained from conventional analyses of MS/MS spectra. The UIS methodology is a novel deterministic peptide identification method for MS/MS spectra based on information content. These robust theoretical assays will have widespread use when integrated with previously collected MS/MS data and conventional proteomics technologies.Shotgun proteomic analyses using multidimensional LC/MS/MS show great capacity for rapid protein analysis. This is arguably the most prevalent work flow for high throughput comparative proteomics, utilizing information-dependent acquisition (IDA)¹ to acquire MS/MS triggered by the signals generated from incoming peptides (1–3). Despite the utility and widespread use of this approach, there remain inherent problems including a relatively high level of ambiguous and false peptide assignments (∼5%) as well as high numbers of unassigned mass spectra (4–6). The reason for this level of ambiguity stems in part from the non-deterministic nature of the identification algorithms. Without the use of reference standards the only way to know a spectrum was generated by a given peptide with absolute certainty is for the spectrum to contain a fragment pattern that conclusively demonstrates the presence of each amino acid. Unfortunately this level of coverage is extremely rare in proteomics data.More recently, selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) mass spectrometry methods have been deployed for proteomic analyses (7–20). This has occurred as proteomics has matured from a discovery-oriented discipline into a more targeted and quantitative field. The method is conventionally conducted using triple quadrupole mass spectrometers where two rounds of mass selection provide excellent fidelity and sensitivity to monitor one or more predetermined target peptides generally in the context of a complex sample such as a cell lysate. Using this approach the mass spectrometer continually monitors the selected precursor ion m/z (Q1) and a subsequent product ion m/z (Q3) from the target analyte. SRM experiments can be used to conduct several rounds of these scans targeting different product ions in an attempt to bolster the confidence that the Q1 → Q3 transitions monitor the intended analyte with fidelity. A key point of contrast with IDA experiments is the need to preselect target analytes for monitoring. This can be achieved by harvesting data from previous discovery-based experiments or by in silico predictions such as MRM-initiated detection and sequencing (MIDAS) (10, 12). Regardless the key underlying principle of SRM in proteomics applications is that the selected set of precursor and product ions contain sufficient information to proxy for the target peptide and thereby its protein of origin. Given that proteomics SRM experiments are conducted with a minimal set of transitions, one must accept that a degree of uncertainty resides in any such assay. To date, the magnitude of this uncertainty has not been studied. This remains a key point even with MS instruments capable of conducting subsequent full MS/MS scans triggered by SRM (e.g. QTrap) as these are lower sensitivity scans that may contain insufficient fragmentation data to conclusively confer peptide identity.The problem of interference is also present in SRM experiments. To achieve acceptable sensitivity a large Q1 m/z window (±0.3–1.0 m/z) is needed. This in turn allows other peptides with similar Q1 m/z and elution properties to interfere with detection of the desired target. The frequency of these interferences would likely increase as the complexity of the sample increases creating a greater likelihood of false positives. Clearly this is not an unexpected result as conventional peptide identification strategies utilizing tandem MS result in some false assignments. Therefore, it would be unreasonable to expect that SRM assays that typically utilize fewer product ions than MS/MS experiments would not also encounter similar interference (21).In this study we investigated the information content of SRM assays and in doing so exposed the potential redundancy. Computational simulations of the experiment enabled us to demonstrate that directed selection of SRM precursor and product ions can avoid the pitfalls of interference by selecting ion combinations that uniquely map to target peptides within the context of the simulation. We used these unique ion signatures (UIS) in a proof of concept study to direct SRM data acquisition for the exclusive detection of enzymes in the Escherichia coli tricarboxylic acid cycle. In addition, given that UIS have been calculated to uniquely define target peptides in the experimental context, we demonstrated the applicability of UIS as an orthogonal validation of peptide identity for traditional MS/MS experiments.     

The pollen virome: A review of pollen-associated viruses and consequences for plants and their interactions with pollinators

The movement of pollen grains from anthers to stigmas, often by insect pollinator vectors, is essential for plant reproduction. However, pollen is also a unique vehicle for viral spread. Pollen-associated plant viruses reside on the outside or inside of pollen grains, infect susceptible individuals through vertical or horizontal infection pathways, and can decrease plant fitness. These viruses are transferred with pollen between plants by pollinator vectors as they forage for floral resources; thus, pollen-associated viral spread is mediated by floral and pollen grain phenotypes and pollinator traits, much like pollination. Most of what is currently known about pollen-associated viruses was discovered through infection and transmission experiments in controlled settings, usually involving one virus and one plant species of agricultural or horticultural interest. In this review, we first provide an updated, comprehensive list of the recognized pollen-associated viruses. Then, we summarize virus, plant, pollinator vector, and landscape traits that can affect pollen-associated virus transmission, infection, and distribution. Next, we highlight the consequences of plant–pollinator–virus interactions that emerge in complex communities of co-flowering plants and pollinator vectors, such as pollen-associated virus spread between plant species and viral jumps from plant to pollinator hosts. We conclude by emphasizing the need for collaborative research that bridges pollen biology, virology, and pollination biology.