CONSTANCY OF RELATIVE VOLUMES OF ZONES IN SHOOT APICAL MERISTEMS IN CACTACEAE: IMPLICATIONS CONCERNING MERISTEM SIZE,SHAPE, AND METABOLISM

The volumes and dimensions of shoot apical meristem zones were determined for 21 species in 10 genera of the Cactaceae. If the volumes of the central-mother-cell zone (CMCZ), the peripheral zone (PZ), and the pith-rib meristem (PRM) are expressed as percentages of the total volume of the corpus, then they are remarkably constant (CMCZ = 11.1% of the volume of the corpus, PZ = 61.2% and PRM = 27.1%) regardless of the relative size of the apical meristems. The relative volume of the tunica decreases, however, as the whole apex increases. The zones have variable shapes, and whereas the PZ and PRM are always trapezoidal in longitudinal section, in apices with large volumes, these zones have a flatter, more flaring shape than they have in smaller apices. Thus large apices are flatter and less hemispherical than are small apices. The CMCZ, however, maintains a constant shape and is always relatively hemispherical regardless of its volume. A hypothesis that relates all of these shape and volume relationships as an integrated growth sequence is proposed.     

CONIFER OVULATE CONE MORPHOLOGY: IMPLICATIONS ON POLLEN IMPACTION PATTERNS

Empirically determined patterns of pollen impaction on the surfaces of pine ovulate cones are correlated with regions of nonlaminar flow created by the spatial arrangement and morphology (aspect ratios) of scale-bract complexes. Results from the serial discharge of pollen, upwind of ovulate cones, indicate that ovules on ovulate cones are preferentially impacted by pollen from their own species. Analyses indicate that while aerodynamic factors dominate the entrapment of pollen by ovulate cones, other factors such as pollen impaction-rebound and rebound-reentrainment are significant. Surface characteristics in addition to the settling velocities of pollen may play important roles in determining pollination efficiency. Wind tunnel analyses of the aerodynamic effects of scale-bract arrangement and aspect ratios indicate that each complex behaves as an aerofoil, deflecting air eddies toward the micropylar ends of ovules. The ovulate cone geometry, as a whole, deflects unidirectional wind into cyclonic vortices around the cone axis, each scale-bract deflecting nonimpacted pollen along orthostichies and parastichies. The morphology of the typical conifer ovulate cone is interpreted as a structure that optimizes anemophilous reproduction.     

Characterization of phase separation in film forming biopolymer mixtures

Enhanced, tailor-made films can be achieved by combining the good gas barrier of the hydrophilic high amylose maize starch (hylon) with the water resistance of the hydrophobic protein zein. Two polymers are not always miscible in solution, and the phase separation behavior of the mixture is therefore important for the final film structure and its properties. Phase separation of a mixture of these two biopolymers was induced either by cooling, which was observed as growing droplets of the hylon phase which in some cases also formed small aggregates, or by solvent evaporation and studied in real-time in a confocal laser scanning microscope. Solvent evaporation had a much stronger effect on phase separation. During the early stage of phase separation, hylon formed large aggregates and subsequently smaller droplets coalesced with other droplets or large hylon aggregates. The later part of the separation seemed to take place through spinodal decomposition.     

Folding into a beta-hairpin can prevent amyloid fibril formation

The tetrapeptide KFFE is one of the shortest amyloid fibril-forming peptides described. Herein, we have investigated how the structural environment of this motif affects polymerization. Using a turn motif (YNGK) or a less rigid sequence (AAAK) to fuse two KFFE tetrapeptides, we show by several biophysical methods that the amyloidogenic properties are strongly dependent on the structural environment. The dodecapeptide KFFEAAAKKFFE forms abundant thick fibril bundles. Freshly dissolved KFFEAAAKKFFE is monomeric and shows mainly disordered secondary structure, as evidenced by circular dichroism, NMR spectroscopy, hydrogen/deuterium exchange measurements, and molecular modeling studies. In sharp contrast, the dodecapeptide KFFEYNGKKFFE does not form fibrils but folds into a stable beta-hairpin. This structure can oligomerize into a stable 12-mer and multiples thereof, as shown by size exclusion chromatography, sedimentation analysis, and electrospray mass spectrometry. These data indicate that the structural context in which a potential fibril forming sequence is present can prevent fibril formation by favoring self-limiting oligomerization over polymerization.     

Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II-related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR-DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.     

Energy Metabolic Changes in the Early Post-injury Period Following Traumatic Brain Injury in Rats

Impaired cerebral energy metabolism may be a major contributor to the secondary injury cascade that occurs following traumatic brain injury (TBI). To estimate the cortical energy metabolic state following mild and severe controlled cortical contusion (CCC) TBI in rats, ipsi-and contralateral cortical tissues were frozen in situ at 15 and 40 min post-injury and adenylate (ATP, ADP, AMP) levels were analyzed using high-performance liquid chromatography (HPLC) and the energy charge (EC) was calculated. At 15 min post-injury, mildly brain-injured animals showed a 43% decrease in cortical ATP levels and a 2.4-fold increase in AMP levels (P < 0.05), and there was a significant reduction of the ipsilateral cortical EC when compared to sham-injured animals (P < 0.05). At 40 min post-injury, the ipsilateral adenylate levels and EC had recovered to the values observed in the sham-injury group. In the severe CCC group, there was a 51% decrease in ipsilateral cortical ATP levels and a 5.3-fold increase in AMP levels with a significant reduction of cortical EC at 15 min post-injury (P < 0.05). At 40 min post-injury, a 2.6-fold ipsilateral increase in AMP levels and an 11% and 44% decrease in EC and ATP levels, respectively, remained (P < 0.05). A 37–38% reduction of the total adenylate pool was observed ipsilaterally in both CCC severity groups at the early time-point, and a 19% and 28% decrease remained in the mild and severe CCC groups, respectively, at 40 min post-injury. Significant contralateral ATP and EC changes were only observed in the severe CCC group at 40 min post-injury (P < 0.05). The energy-requiring secondary injury cascades that occur early post-injury do not challenge the brain tissue to the extent of ATP depletion and may provide a window of opportunity for therapeutic intervention.     

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp-167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro target of AtPDK1. Our data clearly show that Asp-167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident.     

Biomass partitioning and leaf N,P - stoichiometry: comparisons between tree and herbaceous current-year shoots

We compare the biomass partitioning patterns and the nitrogen/phosphorus (N,P) stoichiometry of the current-year shoots of tree and herbaceous species and ask whether they scale in the same ways. Our analyses indicate that few statistically significant differences exist between the shoot biomass partitioning patterns of the two functional species-groups. In contrast, statistically significant N,P - stoichiometric differences exist between the two functional groups. Across all species, dry leaf mass scales nearly as the square of basal stem diameter and isometrically with respect to dry stem mass. However, total leaf N scales as the 1.37-power and as the 1.09-power of total leaf P across herbaceous and tree shoots, respectively. Therefore, tree shoots can be viewed as populations of herbs elevated by their older, woody herbaceous cohorts. However, tree leaf stoichiometry cannot be modelled in terms of herbaceous N,P - leaf stoichiometry.