Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin.

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

The human fetal lymphocyte lineage: identification by CD27 and LIN28B expression in B cell progenitors.

CD27, a member of the TNFR superfamily, is used to identify human memory B cells. Nonetheless, CD27(+) B cells are present in patients with HIGM1 syndrome who are unable to generate GCs or memory B cells. CD27(+)IgD(+) fetal B cells are present in umbilical cord blood, and CD27 may also be a marker of the human B1-like B cells. To define the origin of naïve CD27(+)IgD(+) human B cells, we studied B cell development in both fetal and adult tissues. In human FL, most CD19(+) cells coexpressed CD10, a marker of human developing B cells. Some CD19(+)CD10(+) B cells expressed CD27, and these fetal CD27(+) cells were present in the pro-B, pre-B, and immature/transitional B cell compartments. Lower frequencies of phenotypically identical cells were also identified in adult BM. CD27(+) pro-B, pre-B, and immature/transitional B cells expressed recombination activating gene-1, terminal deoxynucleotidyl transferase and Vpre-B mRNA comparably to their CD27(-) counterparts. CD27(+) and CD27(-) developing B cells showed similar Ig heavy chain gene usage with low levels of mutations, suggesting that CD27(+) developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27(+) developing B cells differed from CD27(-) developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27(+) pro-B cells efficiently generated IgM(+)IgD(+) immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B cell development identifies a physiologic state or lineage for human B cell development distinct from the memory B cell compartment.

Modulation of prothrombinase assembly and activity by phosphatidylethanolamine.

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (K(d) = ∼10 nm) in the absence of phosphatidylserine (PS). These membranes support formation of a 60-70% functional prothrombinase complex at saturating factor Va concentrations. Although reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va(2) (K(d) = ∼6.5 µm) and to factor Xa (K(d) = ∼91 µm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (K(d)(app) = ∼40 nm) of a partially active prothrombinase complex between factor Xa and factor Va(2), compared with K(d)(app) for C6PS ∼2 nm. These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.

Membrane-associated protein kinase activities in the developing mesocarp of grape berry.

Fruit development is a process involving various signals and gene expression. Protein phosphorylation catalyzed by protein kinases is known to play a key role in eukaryotic cell signalling and so may be involved in the regulation of fruit development. Using the method of exogenous substrate phosphorylation, we characterised the calcium-dependent and calmodulin-independent protein kinase (CDPK) activity and the myelin basic protein (MBP)-phosphoralating activity that could be due to a mitogen-activated protein kinase (MAPK)-like activity in the developing mesocarp of grape berry. The CDPK activity was shown to be predominantly localised in the plasma membrane, while the MAPK-like activity was predominantly associated with endomembranes. The assays of bivalent cation requirement showed that Mn2+ could to a certain extent replace Mg2+ in the incubation system for the protein kinase activities. Both CDPK and MAPK-like activities were resistant to heat treatment. The activities of the two enzymes were fruit developmental stage-specific with the highest activities of both enzymes in the lag growth phase before the ripening stage, suggesting strongly the important roles of the detected CDPK and MAPK-like activities in the fruit development.

Molecular cloning of ten distinct hypervariable regions from the cellulose synthase gene superfamily in aspen trees.

Recent molecular genetic data suggest that cellulose synthase (CesA) genes coding for the enzymes that catalyze cellulose biosynthesis (CESAs) in Arabidopsis and other herbaceous plants belong to a large gene family. Much less is known about CesA genes from forest trees. To isolate new CesA genes from tree species, discriminative but easily obtainable homologous DNA probes are required. Hypervariable regions (HVRII) of CesA genes represent highly divergent DNA sequences that can be used to examine structural, expressional and functional relationships among CesA genes. We used a reverse transcriptase-polymerase chain reaction (RT-PCR)-based technique to identify HVRII regions from eight types of CesA genes and two types of CesA-like D (CslD) genes in quaking aspen (Populus tremuloides Michx.). Comparison of these aspen CESA/CSLD HVRII regions with the predicted proteins from eight full-length CesA/CslD cDNAs available in our laboratory and with searches for aspen CesA/CslD homologs in the recently released Populus trichocarpa Torr. & A. Gray. genome confirmed the utility of this approach in identifying several CesA/CslD gene members from the Populus genome. Phylogenetic analysis of 56 HVRII domains from a variety of plant species suggested that at least six distinct classes of CESAs exist in plants, supporting a previous proposal for renaming HVRII regions as class-specific regions (CSR). This method of CSR cloning could be applied to other crop plants and tree species, especially softwoods, for which the whole genome sequence is unlikely to become available in the near future because of the large size of these genomes.

Genomics of cellulose biosynthesis in poplars.

Genetic improvement of cellulose production in commercially important trees is one of the formidable goals of current forest biotechnology research. To achieve this goal, we must first decipher the enigmatic and complex process of cellulose biosynthesis in trees. The recent availability of rich genomic resources in poplars make Populus the first tree genus for which genetic augmentation of cellulose may soon become possible. Fortunately, because of the structural conservation of key cellulose biosynthesis genes between Arabidopsis and poplar genomes, the lessons learned from exploring the functions of Arabidopsis genes may be applied directly to poplars. However, regulation of these genes will most likely be distinct in these two-model systems because of their inherent biological differences. This research review covers the current state of knowledge about the three major cellulose biosynthesis-related gene families from poplar genomes: cellulose synthases, sucrose synthases and korrigan cellulases. Furthermore, we also suggest some future research directions that may have significant economical impacts on global forest product industries.

Identification of GhMYB109 encoding a R2R3 MYB transcription factor that expressed specifically in fiber initials and elongating fibers of cotton (Gossypium hirsutum L.).

Cotton (Gossypium hirsutum L.) fibers are derived from ovule epidermis, which are developmentally similar to Arabidopsis trichome where several MYB transcription factors have been shown to control their formation. However, little is known about the molecular control of cotton fiber initiation. Here we isolated 55 cotton MYB domain-containing sequences expressed in ovules during fiber initiation. Among them, GhMYB109, encoding a R2R3 MYB transcription factor of 234 amino acids, was found to be structurally related to AtMYBGL1 and AtWER controlling the trichome initiation in Arabidopsis thaliana. Southern blot hybridization revealed that GhMYB109 is present as a unique-copy gene in cotton genome. RNA expression analysis showed that it is specifically expressed in cotton fiber initial cells as well as elongating fibers. These results suggested that GhMYB109 likely plays a direct role in the initiation and elongation of cotton fiber cells.

Membrane-associated protein kinase activities in developing apple fruit.

Fruit development is a process involving various signals and gene expression. Protein phosphorylation catalysed by protein kinases is known to play a key role in eukaryotic cell signalling and so may be involved in the regulation of fruit development. Using the method of exogenous substrate phosphorylation, the activity of calcium-dependent and calmodulin-independent protein kinase (CDPK) that was stimulated by phosphatidylserine, and the myelin basic protein (MBP)-phosphorylating activity that could be due to a calcium-independent mitogen-activated protein kinase-like (MAPK-like) activity in the developing apple fruits were identified. The CDPK activity was shown to be predominantly localized in the plasma membrane, whereas in the presence of phosphatidylserine, the high activity of CDPK was detected in both plasma membrane and endomembranes. The MAPK-like activity was predominantly associated with endomembranes. The assays of bivalent cation requirement showed that Mn2+ could replace Mg2+ in the incubation system for the protein kinase activities and stimulate CDPK activity more than Mg2+. Heat treatment abolished CDPK but stimulated MAPK-like activity. The activities of the phosphatidylserine-stimulated CDPK and of the MAPK-like were fruit developmental stage-specific with higher activities of both enzymes in the early and middle developmental stages in comparison with the late developmental stage. These data suggest that the detected protein kinases may play an important role in the fruit development.