Effects of metal-binding loop mutations on ligand binding to calcium- and integrin-binding protein 1. Evolution of the EF-hand?

Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous, multifunctional regulatory protein consisting of four helix-loop-helix EF-hand motifs. Neither EF-I nor EF-II binds divalent metal ions; however, EF-III is a mixed Mg2+/Ca2+-binding site, and EF-IV is a higher-affinity Ca2+-specific site. Through the generation of several CIB1 mutant proteins, we have investigated the importance of the last (-Z) metal-coordinating position of EF-III (D127) and EF-IV (E172) with respect to the binding of CIB1 to Mg2+, Ca2+, and its biological target, the cytoplasmic domain of the platelet alphaIIb integrin. A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain. A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed. E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain. Finally, a D127Q mutant bound Mg2+ and Ca2+ in a manner similar to that of D127N, but like that of D127E, the affinity for the alphaIIb domain was reduced below the detection limit. These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III. Conversely, upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor.     

DEVELOPMENTAL FEATURES OF THE SPERMATOGENOUS CELL IN GINKGO BILOBA

A “double-blepharoplast” originates de novo in the spermatogenous cell of Ginkgo biloba L. Initially, the double-blepharoplast consists of two opposing hemispherical bodies comprised of densely staining material. The two blepharoplasts seemingly are pushed apart by the formation of densely packed fibrils which are oriented perpendicular to the distal, rounded edges of the two future blepharoplasts. As the latter move apart, each one develops lightly staining channels which are often organized in a hub and spoke configuration (procentrioles). Microtubules extend from the blepharoplasts as the latter move to their final position in the cell, and centrioles (probasal bodies) become organized at the periphery of each blepharoplast. Two large “osmiophilic globules,” conspicuous entities close to the nucleus of the mature spermatogenous cell, arise de novo. A fibrillogranular body in the cytoplasm, always closely associated with the nucleus, also arises de novo.     

Litter quality and decomposition in Danthonia richardsonii swards in response to CO2 and nitrogen supply over four years of growth

Litter quality parameters of Danthonia richardsonii grown under CO₂ concentrations of ≈ 359 & ≈ 719 μL L^{? 1} at three mineral N supply rates (2.2, 6.7 & 19.8 g m^{? 2} y^{? 1}) were determined. C:N ratio was increased in senesced leaf (enhancement ratios, R_e/c, of 1.25–1.67), surface litter (1.34–1.64) and root (1.13–1.30) by CO₂ enrichment. After 3 years of growth, nonstructural carbohydrate concentrations were reduced in senesced leaf lamina (avg. R_e/c= 0.84) but not in root in response to CO₂ enrichment. Cellulose concentrations increased slightly in senesced leaf (avg. R_e/c= 1.07) but not in root in response to CO₂ enrichment. Lignin and polyphenolic concentrations in senesced leaf and root were not changed by CO₂ enrichment. Decomposition, measured as cumulative respiration in standard conditions in vitro, was reduced in leaf litter grown under CO₂ enrichment. Root decomposition in vitro was lower in the material produced under CO₂ enrichment at the two higher rates of mineral N supply. Significant correlations between decomposition of leaf litter and initial %N, C:N ratio and lignin:N ratio were found. Decomposition in vivo, measured as carbon disappearance from the surface litter was not affected by CO₂ concentration. Arbuscular mycorrhizal infection was not changed by CO₂ enrichment. Microbial carbon was higher under CO₂ enrichment at the two higher rates of mineral N supply. Possible reasons for the lack of effect of changes in litter quality on in‐sward decomposition rates are discussed.     

A Staining Combination for Phloem and Contiguous Tissues

A technic is described for producing critically stained preparations of phloem tissue. The preparations promise to be relatively stable. Sections of fixed unembedded or of embedded (paraffin or celloidin) phloem, cambium, and xylem are (1) stained in Foster's tannic acid-ferric chloride combination; (2) treated with 1% NaHCOg in 25% or 50% ethyl alcohol for 30 minutes; (3) stained in a saturated solution of lacmoid (made alkaline by adding a few ml. of 1% NaHCO₃ in 25% alcohol) for 12 to 18 hours; (4) dehydrated and cleared in a series composed of 1% solution of NaHCO_s in 50% ethyl alcohol, 80%, 95%, and absolute alcohol, equal proportions of absolute alcohol, clove oil, and xylene, and finally pure xylene; and (5) mounted in a neutral resin. Callose and lignified secondary walls are blue or blue-green in color, cellulose walls and stainable protoplasmic contents are generally light brown. The technic has been successful with sections from 5 to 40μ in thickness, and the staining has been satisfactory for both color and black and white photomicrography.