Flow cytometric analysis of megakaryocyte differentiation

Megakaryocytes were isolated quantitatively from rat bone marrow by centrifugal elutriation (CE). CE-enriched megakaryocytes were stained supravitally for either DNA content with Hoechst 33342, surface membrane immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated antiplatelet antibody, or both. The cells were then measured using a Becton Dickinson FACS IV flow cytometer. The following correlations were analyzed: DNA content and light scatter, light scatter and antiplatelet immunofluorescence, and DNA content and antiplatelet immunofluorescence. Although the range of light scatter increased as a function of DNA content, discrete subpopulations of megakaryocytes with different light scatter properties were detected within each of the three principal ploidy classes (8C, 16C, and 32C). Other discrete megakaryocyte subpopulations were revealed in the analysis of antiplatelet surface immunofluorescence as a function of degree of light scatter. The nonlinear relationship between the latter suggested that the degree of membrane immunofluorescence did not bear a simple relationship to cell size as reflected in light scatter. Megakaryocyte DNA content, on the other hand, varied in a linear fashion with membrane immunofluorescence, supporting the conclusion that there may be a proportional increase in the expression of platelet antigens with DNA content. The use of multiple markers, correlated multiparameter flow cytometry and multivectorial analysis to define differentiation on a single cell basis have revealed new complexities in this process. Flow cytometric analysis holds promise as a useful method for further characterization of megakaryocyte differentiation.     

Glutathione reductase from human erythrocytes. Molecular weight, subunit composition and aggregation properties.

Glutathione reductase from human erythrocytes exists predominatly as an entity of 100 000 molecular weight under various conditions of pH and ionic strength. The S20,W of 5.5 S and D20W of 50 mum2/s correlate with the molecular weight determined by sedimentation equilibrium. The homogeneity of this species is primarily dependent on the presence of thiols and secondarily on high concentrations of salt. The amino-acid composition of the enzyme shows similarities both with glutathione reductases from other sources and with lipoamide dehydrogenase. From the flavin content and dodecylsulphate-polyacrylamide electrophoresis it is inferred that the native enzyme is a dimer composed of similar subunits of 50 000 molecular weight. In the absence of thiols, glutathione reductase shows a tendency to form tetramers and larger aggregates. Although these larger species are also catalytically active, under cellular conditions the presence of its product, reduced glutathione, should maintain the enzyme as the dimeric entity.     

Low angle x-ray diffraction and biology

The use of the low-angle x-ray diffraction method in biology is described. Some x-ray diffraction patterns have been known since the early 1930's but, in the last decade, many new patterns, which show more reflections than previously, have been recorded. An interpretation of these improved patterns of biological systems provides detailed information on the molecular structure of living cells. The theory and the current status of the low-angle x-ray diffraction method is presented. A brief account of the x-ray studies on collagen, muscle, nerve and membranes is given. Emphasis is given to the history of ideas underlying progress in this area and the current unsolved problems in this field of biophysical research are indicated and discussed.     

Spatial distribution and prediction of seed production by Eucalyptus microcarpa in a fragmented landscape

Woodlands worldwide have been greatly modified by clearing for agriculture, and their conservation and restoration requires understanding of tree recruitment processes. Seed production is one possible point of recruitment failure, and one that the spatial arrangement of trees may affect. We sampled 118 Eucalyptus microcarpa (Myrtaceae) trees to compare and analyse the determinants of seed production in this dominant tree of modified, fragmented temperate grassy woodlands, which extend over much of southeastern Australia. Fecundity was estimated as the seed crop measured on leaf mass and whole tree bases and was compared between categories of tree configuration. We also modelled fecundity using boosted regression trees, a new and flexible tool. Fecundity on a leaf mass basis was predominantly influenced by environmental factors (topographic ‘wetness’, slope, soil type), rather than by local tree density and configuration. Fewer seed per unit leaf mass were produced on flat and topographically wet sites, reflecting poor tolerance of waterlogging by E. microcarpa. By contrast, whole tree fecundity was little influenced by environmental factors. Local tree density and configuration did influence whole tree fecundity, which was high in solitary and woodland‐spaced trees and reduced under high local density. We found little evidence for reduced fecundity of E. microcarpa in solitary trees. This points to the importance of scattered trees as sources of seed for tree recruitment and for natural regeneration of landscape level tree cover. Considerable uncertainty remains in modelled seed supply, and may be reduced with sampling across multiple years and greater environmental and spatial domains.