Display of a Maize cDNA library on baculovirus infected insect cells

Background  

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings.     

Bridging “Romer’s Gap”: Limb Mechanics of an Extant Belly-Dragging Lizard Inform Debate on Tetrapod Locomotion During the Early Carboniferous

Devonian stem tetrapods are thought to have used ‘crutching’ on land, a belly-dragging form of synchronous forelimb action-powered locomotion. During the Early Carboniferous, early tetrapods underwent rapid radiation, and the terrestrial locomotion of crown-group node tetrapods is believed to have been hindlimb-powered and ‘raised’, involving symmetrical gaits similar to those used by modern salamanders. The fossil record over this period of evolutionary transition is remarkably poor (Romer’s Gap), but we hypothesize a phase of belly-dragging sprawling locomotion combined with symmetrical gaits. Since belly-dragging sprawling locomotion has differing functional demands from ‘raised’ sprawling locomotion, we studied the limb mechanics of the extant belly-dragging blue-tongued skink. We used X-ray reconstruction of moving morphology to quantify the three-dimensional kinematic components, and simultaneously recorded single limb substrate reaction forces (SRF) in order to calculate SRF moment arms and the external moments acting on the proximal limb joints. In the hindlimbs, stylopodal long-axis rotation is more emphasized than in the forelimbs, and much greater vertical and propulsive forces are exerted. The SRF moment arm acting on the shoulder is at a local minimum at the instant of peak force. The hindlimbs display patterns that more closely resemble ‘raised’ sprawling species. External moment at the shoulder of the skink is smaller than in ‘raised’ sprawlers. We propose an evolutionary scenario in which the locomotor mechanics of belly-dragging early tetrapods were gradually modified towards hindlimb-powered, raised terrestrial locomotion with symmetrical gait. In accordance with the view that limb evolution was an exaptation for terrestrial locomotion, the kinematic pattern of the limbs for the generation of propulsion preceded, in our scenario, the evolution of permanent body weight support.     

Competition between bacteriophage f2 RNA and bacteriophage T4 messenger RNA

In an $\text{Escherichia coli}$ cell-free protein synthesis assay, mRNA isolated from cells late after infection by phage T4 out-competes bacteriophage f2 RNA. Addition of a saturating or subsaturating amount of T4 mRNA inhibits translation of f2 RNA, while even an excess of f2 RNA has no effect on translation of T4 mRNA. Peptide mapping of reaction products labeled with formyl-[³⁵S]-methionyl-tRNA was used to quantitate f2 and T4 protein products synthesized in the same reaction. We suggest that messenger RNA competition might be one mechanism by which T4 superinfection of cells infected with phage f2 blocks translation of f2 RNA and possibly host mRNA.     

Cytogenetic analysis of three sea catfish species (Teleostei, Siluriformes, Ariidae) with the first report of Ag-NOR in this fish family

Despite their ecological and economical importance, fishes of the family Ariidae are still genetically and cytogenetically poorly studied. Among the 133 known species of ariids, only eight have been karyotyped. Cytogenetic analyses performed on Genidens barbus and Sciades herzbergii revealed that both species have 2n = 56 chromosomes and Cathorops aff. mapale has 2n = 52 chromosomes: Genidens barbus has 10 Metacentrics (M), 14 Submetacentrics (SM), 26 Subtelocentrics (ST), and 6 Acrocentrics (A), Sciades herzbergii has 14M, 20SM, 18ST and 4A, whereas Cathorops aff. mapale has 14M, 20SM, and 18ST. The nucleolus organizer regions (NORs) were found in a single chromosome pair on the short arm of a large-sized ST pair in Genidens barbus and on the short arm of a middle-size SM pair in Cathorops aff. mapale. Multiple NORs on the short arms of two large-sized ST pairs were found in Sciades herzbergii. The occurrence of diploid numbers ranging from 2n = 52 through 56 chromosomes and the presence of different karyotypic compositions, besides the number and position of NORs suggest that several numeric and structural chromosome rearrangements were fixed during the evolutionary history of this fish family.     

Bacillus anthracis Spore Entry into Epithelial Cells Is an Actin-Dependent Process Requiring c-Src and PI3K

Dissemination of Bacillus anthracis from the respiratory mucosa is a critical step in the establishment of inhalational anthrax. Recent in vitro and in vivo studies indicated that this organism was able to penetrate the lung epithelium by directly entering into epithelial cells of the lung; however the molecular details of B. anthracis breaching the epithelium were lacking. Here, using a combination of pharmacological inhibitors, dominant negative mutants, and colocalization experiments, we demonstrated that internalization of spores by epithelial cells was actin-dependent and was mediated by the Rho-family GTPase Cdc42 but not RhoA or Rac1. Phosphatidylinositol 3-kinase (PI3K) activity was also required as indicated by the inhibitory effects of PI3K inhibitors, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, and a PI3K dominant negative (DN) mutant Δp85α. In addition, spore entry into epithelial cells (but not into macrophages) required the activity of Src as indicated by the inhibitory effect of Src family kinase (SFK) inhibitors, PP2 and SU6656, and specific siRNA knockdown of Src. Enrichment of PI3K and F-actin around spore attachment sites was observed and was significantly reduced by treatment with SFK and PI3K inhibitors, respectively. Moreover, B. anthracis translocation through cultured lung epithelial cells was significantly impaired by SFK inhibitors, suggesting that this signaling pathway is important for bacterial dissemination. The effect of the inhibitor on dissemination in vivo was then evaluated. SU6656 treatment of mice significantly reduced B. anthracis dissemination from the lung to distal organs and prolonged the median survival time of mice compared to the untreated control group. Together these results described a signaling pathway specifically required for spore entry into epithelial cells and provided evidence suggesting that this pathway is important for dissemination and virulence in vivo.     

Genetic and Phenotypic Characterization of GII-4 Noroviruses That Circulated during 1987 to 2008

The predominance and continual emergence of new variants in GII-4 noroviruses (NVs) in recent years have raised questions about the role of host immunity and histo-blood group antigens (HBGAs) in NV evolution. To address these questions, we performed a genetic and phenotypic characterization of GII-4 variants circulating in the past decade (1998 to 2008). Ninety-three GII-4 sequences were analyzed, and of them, 16 strains representing 6 genetic clusters were selected for further characterization. The HBGA binding properties were determined by both saliva- and oligosaccharide-binding assays using P particles as a model of NV capsid. The antigenic properties were also examined by enzyme immunoassay (EIA), Western blot analysis, and receptor blocking assay, using P-particle-specific antibodies from immunized mice and GII-4 virus-infected patients. Our results showed that 15 of the 16 GII-4 viruses bound to saliva of all A, B, and O secretors. Oligosaccharide binding assays yielded largely consistent results, although the binding affinities to some oligosaccharides varied among some strains. The only nonbinder had a mutation in the binding site. While antigenic variations were detected among the 16 strains, significant cross-blocking on the HBGA binding was also noted. Sequence alignment revealed high conservation of HBGA binding interfaces with some variations in adjacent regions. Taken together, our data suggested that the ability of GII-4 to recognize different secretor HBGAs persisted over the past decade, which may explain the predominance of GII-4 over other genotypes. Our data also indicated that both the host immunity and HBGAs play a role in NV evolution. While host immunity may continue driving NV for antigenic change, the functional selection by the HBGAs tends to lock the architecture of the capsid/HBGA interfaces and allows only limited variations outside the HBGA binding sites. A potential outcome of such counterselection between theses two factors in NV evolution is discussed.Noroviruses (NVs) have been recognized as the most important cause of nonbacterial acute gastroenteritis in both developed and developing countries, affecting people of all ages (13, 35, 39, 44, 48, 56). They are single-stranded positive-sense RNA viruses belonging to the family Caliciviridae. NVs are highly contagious, spreading by a fecal/oral pathway through person-to-person contact and by contaminated food and/or water and usually causing large outbreaks within closed communities in a variety of settings, such as hospitals, nursing homes, schools, childcare centers, restaurants, cruise ships, and the military (11, 63). Human NVs have been difficult to study due to diverse members and the lack of an efficient cell culture and animal model for human NVs. The cloning of the NV genomes (33, 36, 73) and subsequent expression of the viral capsid proteins in baculovirus and other expression systems (3, 31, 32) have greatly advanced the research of NVs, including host-virus interaction, immunology, diagnosis, molecular virology, and epidemiology (16, 17, 19, 20, 25, 28-30, 46, 51, 59, 73).Several lines of evidence indicate that NVs recognize human histo-blood group antigens (HBGAs) as a ligand or receptor in a strain-specific manner (63, 64). HBGAs are complex carbohydrates presenting on red blood cells and on the epithelia of digestive, respiratory, and genitourinary tracts. They also exist in biologic fluid, such as milk and saliva. NVs are highly diverse in recognizing the human HBGAs, and a number of HBGA-binding patterns involving the ABO, secretor, and Lewis families of human HBGAs have been described (19, 20, 23, 24, 26, 28, 43, 45, 55). The association of HBGA binding with clinical infection and illness has been demonstrated by volunteer challenge studies and outbreak investigations (25, 27, 42, 62, 66), although exceptions also have been reported (41, 50, 53). Further study has mapped the HBGA binding site in the protruding (P) domain of the viral capsid protein (60). Using the P domain as a model, the atomic structures of the HBGA binding interfaces have been resolved by X-ray crystallography (5, 7, 9). The interfaces are comprised of several amino acids located on the top of the P dimer, within the outermost surface of the viral capsid. Extensive hydrogen bond networks between the P dimer and the HBGAs were elucidated and further confirmed by mutagenesis analyses (61, 68, 69). Despite significant differences in genetics and HBGA binding patterns, the sequences of the HBGA-binding interfaces are highly conserved within, but not between, the two major human-related genogroups (GI and GII) of NVs, suggesting that HBGAs are important factors in NV evolution (9, 69).The NV capsid is composed of a single major structural protein, the capsid protein (VP1), which can be divided into two major domains: the shell (S) and the protruding (P) domains (52). Expression of the full-length VP1 by a eukaryotic system forms empty virus-like particles (VLPs) that have been used as a surrogate for NVs for many years, e.g., in diagnostic tests. Recent studies showed that expression of the P domain alone results in the formation of a subviral particle, the P particle (54, 60). Owing to its easy production in an Escherichia coli system and the same HBGA-binding properties and antigenicity as its parental VLP, the P particle has been used as a research tool of NV-HBGA interaction in a number of studies (54, 59, 60, 67, 68, 69). This report took advantage of the convenient P particle model to study the phenotypic HBGA-binding properties and antigenicity of GII-4 NVs that have circulated in the past decade.NVs are grouped into five genogroups (GI to GV), of which GI and GII are involved in the majority of acute viral gastroenteritis cases in humans. Strains within each genogroup can be further divided into genotypes, and up to 30 genotypes of GI and GII NVs have been described (75). NVs can be detected throughout the year, with peaks during the fall and winter seasons. Strains representing multiple genotypes can be found cocirculating in the same geographical area during a season. However, a single genotype of NVs, GII-4 (genogroup II genotype 4), has been the predominant cause of major acute gastroenteritis epidemics in many countries since the mid-1990s, and the number of GII-4 epidemics has increased in recent years (49). Overall, the GII-4 genotype is estimated to be responsible for 60 to 80% of all NV-associated outbreaks worldwide (43).Molecular surveillance has found that the GII-4 viruses are continuously changing, with new variants emerging every 2 or 3 years (1, 2, 57, 71, 72). One hypothesis suggests that the GII-4 viruses might be under selection pressure of the herd immunity, similar to the epochal evolution model used to describe the evolution of influenza (flu) viruses (56). New antigenic variants of GII-4 derived by genetic shift (replacement) accompanied by changes of HBGA binding specificities have been reported (43). However, the HBGA-binding interfaces of NVs have been found to be highly conserved among NVs within each of the two major genogroups, supporting HBGAs as an important factor in NV evolution (69). In fact, it has been shown that the major HBGA-binding pattern of GII-4 viruses to the H3, Le^b, and Le^y antigens has remained unchanged from 1974 to 1997 (4, 23, 24).The objective of this study was to elucidate the roles of HBGAs and host immunity in NV evolution using GII-4 viruses as a model. Since most of the studies on the epochal evolution of GII-4 were based on genetic analysis and focused on GII-4 variants identified in the past decade, we performed a study on the GII-4 variants in the same period by both genetic and phenotypic characterizations. Phylogenetic analysis revealed 6 genetic clusters of GII-4 viruses similar to those reported before. Characterization of HBGA-binding patterns of the GII-4 viruses revealed a consensus phenotype of binding to all A, B, and O secretor HBGAs, with some variations in affinity to these antigens. We also discussed the role of both host immunity and HBGAs in NV evolution. While the host immunity may drive NVs for change, as a functional selection factor, the HBGAs may restrict variation. This counterselection mechanism may help in understanding the epochal evolution hypothesis. The principles found through the study of GII-4 NVs can also be applied to other genotypes, which may eventually lead to a refined functional classification of all NVs.     

Valinomycin-proton interaction in low-polarity media

Valinomycin is shown to form a stable complex with protons in the form of H3O+ ions. Using 1H and 13C nuclear magnetic resonance (NMR), Fourier transform (FT) infrared spectroscopy, and ab initio-density functional theory (DFT) quantum mechanical calculations, it is shown that H3O+, produced by hydrogen bis(1,2-dicarbollyl) cobaltate (HDCC) in the presence of water, interacts with valinomycin in 1,1,2,2-tetrachloroethane-d2 to give a relatively stable complex. The equilibrium constant K of the complex formation was derived from chemical shifts in 1H-NMR, its value being 5.9. The proton affinity constant estimated from this value taking into account HDCC dissociation is 10(5.3), i.e., high enough to be relevant under physiological conditions. Under suitable conditions there is a fast exchange of H3O+ between valinomycin molecules, the exchange correlation time being of the order of 10(-4) s. The instantaneous structure of the complex is slightly asymmetric, the H3O+ ion being strongly hydrogen-bonded to three of the six ester carbonyl groups and weekly bound to the residual ones by electrostatic interactions. This asymmetry is averaged by fast reorientation and displacement of H3O+ so that the molecule appears to be symmetric in the time window of NMR. The results indicate that valinomycin could serve as a carrier for proton transfer across a biological membrane.