Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Oriented thick and thin filaments in amoeba proteus

Actin and myosin filaments as a foundation of contractile systems are well established from ameba to man (3). Wolpert et al. (19) isolated by differential centrifugation from Amoeba proteus a motile fraction composed of filaments which moved upon the addition of ATP. Actin filaments are found in amebas (1, 12, 13) which react with vertebrate heavy meromyosin (HMM), forming arrowhead complexes as vertebrate actin (3, 9), and are prominent within the ectoplasmic tube where some of them are attached to the plasmalemma (1, 12). Thick and thin filaments possessing the morphological characteristics of myosin and actin have been obtained from isolated ameba cytoplasm (18, 19). In addition, there are filaments exhibiting ATPase activity in amebas which react with actin (12, 16, 17). However, giant ameba (Chaos-proteus) shapes are difficult to preserve, and the excellent contributions referred to above are limited by visible distortions occurring in the amebas (rounding up, pseudopods disappearing, and cellular organelles swelling) upon fixation. Achievement of normal ameboid shape in recent glycerination work (15) led us to attempt other electron microscope fixation techniques, resulting in a surprising preservation of A. proteus with a unique orientation of thick and thin filaments in the ectoplasmic region.     

A FINE STRUCTURAL INVESTIGATION OF SURFACE SPECIALIZATIONS AND THE CORTICAL REACTION IN EGGS OF THE CNIDARIAN BUNODOSOMA CAVERNATA

Developing oocytes of the cnidarian Bunodosoma cavernata are located within the mesoglea of the mesenteries of the gastrovascular cavity. The cortex of the more mature vitellogenic oocytes contains numerous, electron-dense, membrane-bound, cortical granules. The surface of these oocytes possesses prominent radially projecting structures termed cytospines. Each cytospine has a core of microfilaments, 50–70 Å in diameter, that extends basally as a rootlet through the cortical layer. During spawning, ova lacking any extraneous investments are released from the enclosing gastrodermis. As a consequence of fertilization or events associated with the earliest stages of development the ova undergo a massive cortical reaction. This reaction, which occurs during or just after release of the ova, involves extensive reorganization of the cortical layer. The cortical granule membranes and egg surface membrane fuse and vesiculate resulting in the massive discharge of granule contents. This event is accompanied by the loss of vesicular remnants of cortical ooplasm and the disruption of cytospine organization. Light and electron microscope comparisons of unreacted and reacted eggs show that the reaction results in a significant decrease in egg diameter with the oolemma of the reacted egg reorganizing in a position centripetal to its original location.     

Studies on the tardigrades. I. Fine structure of the anterior foregut of Milnesium tardigradum Doyère

The fine structure of the anterior foregut of the tardigrade Milnesium tardigradum is presented. The oral region consists of a terminal mouth opening surrounded by six plate-like lips lying within a circlet of six prominent papillae. The buccal cavity is enclosed within a thick cuticular tube which possesses appendage structures, the stylet sheaths, stylet supports and paired protrusible stylets. Two large salivary glands envelop the buccal structures and contain voluminous amounts of secretory product. The arrangement, possible functions and phyletic significance of these structures are discussed.     

Phylogenetic analyses of the rbcL sequences from haptophytes and heterokont algae suggest their chloroplasts are unrelated

Using the large subunit of RuBisCo (rbcL) sequences from cyanobacteria, proteobacteria, and diverse groups of algae and green plants, we evaluated the plastid relationship between haptophytes and heterokont algae. The rbcL sequences were determined from three taxa of heterokont algae (Bumilleriopsis filiformis, Pelagomonas calceolata, and Pseudopedinella elastica) and added to 25 published sequences to obtain a data set comprising 1,434 unambiguously aligned sites (approximately 98% of the total rbcL gene). Higher levels of mutational saturation in third codon positions were observed by plotting the pairwise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In accordance with this finding phylogeny reconstructions were completed by omitting third codon positions, thus using 956 bp in weighted-parsimony and maximum-likelihood analyses. The midpoint-rooted phylogenies showed two major clusters, one containing cyanobacteria, glaucocystophytes, a phototrophic euglenoid, chlorophytes, and embryophytes (the green lineage), the other containing proteobacteria, haptophytes, red algae, a cryptophyte, and heterokont algae (the non-green lineage). In the nongreen lineage, the haptophytes formed a sister group to the clade containing heterokont algae, red algae, and the cryptophyte Guillardia theta. This branching pattern was well supported in terms of bootstrap values in weighted- parsimony and maximum-likelihood analyses (100% and 92%, respectively). However, the phylogenetic relationship among red algae, heterokonts, and a cryptophyte taxon was not especially well resolved. A four- cluster analysis was performed to further explore the statistical significance of the relationship between proteobacteria, red algae (including and excluding Guillardia theta), haptophytes, and heterokont algae. This test strongly favored the hypothesis that the heterokonts and red algae are more closely related to each other than either is to proteobacteria or haptophytes. Hence, this molecular study based on a plastid-encoded gene provides additional evidence for a distant relationship between haptophytes and the heterokont algae. It suggests an evolutionary scenario in which the ancestor of the haptophyte lineage engulfed a phototrophic eukaryote and, more recently, the heterokont lineage became phototrophic by engulfing a red alga.      

Variation in the harvest index of tropical maize: evaluation of recent evidence from Mexico and Malawi

In temperate zones, the potential grain yield of wheat has increased during the twentieth century owing to progressive increases in the harvest index of new varieties, which are principally associated with reduction in plant stature. Crop biomass has not increased substantially. In contrast, the potential grain yield of maize in the USA has increased owing to progressive increases in biomass, principally associated with selection for grain yield at higher population density. Harvest index was already around 0.5 for recommended varieties in 1930, and has not increased significantly since. However, for both crops, the harvest index of a given variety has proved to be a highly‐heritable character, except under severe stress. Less is known about the physiology of tropical maize. This paper reviews evidence from Mexico and Malawi that tropical maize can respond to selection for reduced stature following the same pattern as temperate wheat, but, under other circumstances, the magnitude of harvest index is not highly heritable, varying inconsistently with season, management and environment. It is proposed that these differences arise out of the unique vulnerability of the grain‐setting process around flowering. The plasticity of harvest index under long, favourable conditions, however, remains to be explained, although it is probably also related to the events around grain setting. Nevertheless, to the subsistence farmer, higher harvest index may not be a high priority in crop improvement, because of the need for large quantities of high‐quality stover.     

Morphological Constraints of Shoot Demography of a Clonal Plant: Extra- and Intravaginal Tillers of Festuca rubra

Abstract Festuca rubra forms tillers in two different ways: extravaginally and intravaginally. Demography of these two tiller types was observed in seventeen selected tussocks of Festuca rubra s.s. over four growing seasons. Extravaginal tillers were bigger at birth and on the average produced twice as many daughter tillers per tiller. In general, the natality and mortality of extravaginal tillers were less regular than that of intravaginal tillers. Overall tillering rate per tiller was correlated with the density of the surrounding vegetation; mortality, natality and tiller life span were not. High density of the surrounding vegetation did not result in increased formation of extravaginal tillers. The proportion of the extravaginal tillers was not correlated with the density of the F. rubra tussocks. There is no evidence for foraging by extravaginal tillers, but they do act as founders of small clusters of tillers.     

Distribution and conservation of mobile elements in the genus Drosophila

Essentially nothing is known of the origin, mode of transmission, and evolution of mobile elements within the genus Drosophila. To better understand the evolutionary history of these mobile elements, we examined the distribution and conservation of homologues to the P, I, gypsy, copia, and F elements in 34 Drosophila species from three subgenera. Probes specific for each element were prepared from D. melanogaster and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the similarity between D. melanogaster sequences and their homologues in other species. The I element homologues show the most limited distribution of all elements tested, being restricted to the melanogaster species group. The P elements are found in many members of the subgenus Sophophora but, with the notable exception of D. nasuta, are not found in the other two subgenera. Copia-, gypsy-, and F-element homologues are widespread in the genus, but their similarity to the D. melanogaster probe differs markedly between species. The distribution of copia and P elements and the conservation of the gypsy and P elements is inconsistent with a model that postulates a single ancient origin for each type of element followed by mating-dependent transmission. The data can be explained by horizontal transmission of mobile elements between reproductively isolated species.