Emission of CO2 and N2O from soil cultivated with common bean (Phaseolus vulgaris L.) fertilized with different N sources.

Addition of different forms of nitrogen fertilizer to cultivated soil is known to affect carbon dioxide (CO(2)) and nitrous oxide (N(2)O) emissions. In this study, the effect of urea, wastewater sludge and vermicompost on emissions of CO(2) and N(2)O in soil cultivated with bean was investigated. Beans were cultivated in the greenhouse in three consecutive experiments, fertilized with or without wastewater sludge at two application rates (33 and 55 Mg fresh wastewater sludge ha(-1), i.e. 48 and 80 kg N ha(-1) considering a N mineralization rate of 40%), vermicompost derived from the wastewater sludge (212 Mg ha(-1), i.e. 80 kg N ha(-1)) or urea (170 kg ha(-1), i.e. 80 kg N ha(-1)), while pH, electrolytic conductivity (EC), inorganic nitrogen and CO(2) and N(2)O emissions were monitored. Vermicompost added to soil increased EC at onset of the experiment, but thereafter values were similar to the other treatments. Most of the NO(3)(-) was taken up by the plants, although some was leached from the upper to the lower soil layer. CO(2) emission was 375 C kg ha(-1) y(-1) in the unamended soil, 340 kg C ha(-1) y(-1) in the urea-amended soil and 839 kg ha(-1) y(-1) in the vermicompost-amended soil. N(2)O emission was 2.92 kg N ha(-1) y(-1) in soil amended with 55 Mg wastewater sludge ha(-1), but only 0.03 kg N ha(-1) y(-1) in the unamended soil. The emission of CO(2) was affected by the phenological stage of the plant while organic fertilizer increased the CO(2) and N(2)O emission, and the yield per plant. Environmental and economic implications must to be considered to decide how many, how often and what kind of organic fertilizer could be used to increase yields, while limiting soil deterioration and greenhouse gas emissions.

Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergent extension.

We have characterized the cell movements and prospective cell identities as neural folds fuse during neural tube formation in Xenopus laevis. A newly developed whole-mount, two-color fluorescent RNA in situ hybridization method, visualized with confocal microscopy, shows that the dorsal neural tube gene xpax3 and the neural-crest-specific gene xslug are expressed far lateral to the medial site of neural fold fusion and that expression moves medially after fusion. To determine whether cell movements or dynamic changes in gene expression are responsible, we used low-light videomicroscopy followed by fluorescent in situ and confocal microscopy. These methods revealed that populations of prospective neural crest and dorsal neural tube cells near the lateral margin of the neural plate at the start of neurulation move to the dorsal midline using distinctive forms of motility. Before fold fusion, superficial neural cells apically contract, roll the neural plate into a trough and appear to pull the superficial epidermal cell sheet medially. After neural fold fusion, lateral deep neural cells move medially by radially intercalating between other neural cells using two types of motility. The neural crest cells migrate as individual cells toward the dorsal midline using medially directed monopolar protrusions. These movements combine the two lateral populations of neural crest into a single medial population that form the roof of the neural tube. The remaining cells of the dorsal neural tube extend protrusions both medially and laterally bringing about radial intercalation of deep and superficial cells to form a single-cell-layered, pseudostratified neural tube. While ours is the first description of medially directed cell migration during neural fold fusion and re-establishment of the neural tube, these complex cell behaviors may be involved during cavitation of the zebrafish neural keel and secondary neurulation in the posterior axis of chicken and mouse.

Measurements of mechanical properties of the blastula wall reveal which hypothesized mechanisms of primary invagination are physically plausible in the sea urchin Strongylocentrotus purpuratus.

Computer simulations showed that the elastic modulus of the cell layer relative to the elastic modulus of the extracellular layers predicted the effectiveness of different force-generating mechanisms for sea urchin primary invagination [L. A. Davidson, M. A. R. Koehl, R. Keller, and G. F. Oster (1995) Development 121, 2005-2018]. Here, we measured the composite elastic modulus of the cellular and extracellular matrix layers in the blastula wall of Strongylocentrotus purpuratus embryos at the mesenchyme blastula stage. Combined, these two layers exhibit a viscoelastic response with an initial stiffness ranging from 600 to 2300 Pa. To identify the cellular structures responsible for this stiffness we disrupted these structures and correlated the resulting lesions to changes in the elastic modulus. We treated embryos with cytochalasin D to disrupt the actin-based cytoskeleton, nocodazole to disrupt the microtubule-based cytoskeleton, and a gentle glycine extraction to disrupt the apical extracellular matrix (ECM). Embryos treated less than 60 min in cytochalasin D showed no change in their time-dependent elastic modulus even though F-actin was severely disrupted. Similarly, nocodazole had no effect on the elastic modulus even as the microtubules were severely disrupted. However, glycine extraction resulted in a 40 to 50% decrease in the elastic modulus along with a dramatic reduction in the hyalin protein at the apical ECM, thus implicating the apical ECM as a major mechanical component of the blastula wall. This finding bears on the mechanical plausibility of several models for primary invagination.

Surface mesoderm in Xenopus: a revision of the stage 10 fate map.

We have used two complementary cell labeling techniques to investigate dorsal mesoderm formation in Xenopus laevis and Hymenochirus boettgeri. Epithelial grafts from fluorescently labeled donors into unlabeled hosts demonstrate that in Xenopus, as previously shown for Hymenochirus, superficial cells of the dorsal marginal zone have the ability to invade the notochord and somite and participate in their normal morphogenesis, in a stage-specific and region-specific manner. A new method for superficial fate mapping using cell surface biotinylation confirms this result for Hymenochirus and demonstrates that in Xenopus as well, even in normal development in the absence of surgical disruption, notochord and the most posterior somitic mesoderm originate partly in the superficial epithelial layer. This finding is contrary to the widespread belief that Xenopus mesoderm originates solely in the deep mesenchymal layer. In Xenopus (but not in Hymenochirus), the amount of superficial contribution to mesoderm varies, such that in some spawnings it appears not to be present, while in others it is evident in all or most embryos.

Fibronectin, mesoderm migration, and gastrulation in Xenopus.

The role of fibronectin in mesoderm cell migration and and the importance of mesoderm migration for gastrulation in Xenopus are examined. To allow for migration, a stable interface must exist between migrating mesoderm cells and the cells of the substrate layer, the blastocoel roof. We show that maintenance of this interface does not depend on fibronectin. We further demonstrate that fibronectin contributes to, but is not essential for, mesoderm cell adhesion to the blastocoel roof. However, interaction with fibronectin is necessary for cell spreading and the formation of lamelliform cytoplasmic protrusions. Apparently, the specific role of fibronectin in mesoderm migration is to control cell protrusive activity. Consequently, when fibronectin function is blocked by GRGDSP peptide or antibodies, mesoderm cell migration is inhibited. Nevertheless, gastrulation proceeds nearly normally in inhibitor-treated embryos. It appears that in Xenopus, mesoderm migration is not essential for gastrulation.

Dorsal mesoderm has a dual origin and forms by a novel mechanism in Hymenochirus, a relative of Xenopus.

The dorsal mesoderm in the frog Hymenochirus forms by a mechanism not previously described in any other vertebrate. Unlike its close relative, Xenopus laevis, in which the mesoderm derives entirely from the deep mesenchymal cells of the marginal zone, Hymenochirus has "surface mesoderm" originating in the involuting marginal zone epithelium. Fluorescently labeled grafts show stage-specific invasion of deep axial tissue by cells originally located in the surface layer. These cells participate in normal mesoderm development. In video recordings, the labeled surface area shrinks as surface cells invade the deep layer. Furthermore, the mechanism of surface mesoderm morphogenesis differs from that described in other amphibians. Scanning electron microscopy at several neurula stages indicates that prospective somite cells do not individually detach from their epithelial neighbors to ingress into the deep layer, as seen in other amphibians; instead, their basal ends adhere to the somitic mesoderm as a coherent layer, taking on somitic morphology while still a part of the archenteron lining. This novel morphogenetic process we dub "relamination." Prospective notochord cells individually spread on the ventral surface of the notochord, gradually ingressing from their epithelial neighbors, but by a mechansism involving active pulling and spreading by their invasive basal ends rather than depending on apical constriction as do the corresponding "bottle cells" in other amphibians. Lateral endoderm migrates dorsally, replacing the relaminating surface mesoderm and fusing at the dorsal midline of the archenteron. These processes demonstrate the diversity of morphogenesis at the cellular, and presumably the molecular, level and shed light on the evolution of morphogenetic mechanisms.

The dorsal involuting marginal zone stiffens anisotropically during its convergent extension in the gastrula of Xenopus laevis.

Physically, the course of morphogenesis is determined by the distribution and timing of force production in the embryo and by the mechanical properties of the tissues on which these forces act. We have miniaturized a standard materials-testing procedure (the stress-relaxation test) to measure the viscoelastic properties of the dorsal involuting marginal zone, prechordal mesoderm, and vegetal endoderm of Xenopus laevis embryos during gastrulation. We focused on the involuting marginal zone, because it undergoes convergent extension (an important and wide-spread morphogenetic process) and drives involution, blastopore closure and elongation of the embryonic axis. We show that the involuting marginal zone stiffens during gastrulation, stiffening is a special property of this region rather than a general property of the whole embryo, stiffening is greater along the anteroposterior axis than the mediolateral axis and changes in the cytoskeleton or extracellular matrix are necessary for stiffening, although changes in cell-cell adhesions or cell-matrix adhesions are not ruled out. These findings provide a baseline of data on which future experiments can be designed and make specific, testable predictions about the roles of the cytoskeleton, extracellular matrix and intercellular adhesion in convergent extension, as well as predictions about the morphogenetic role of convergent extension in early development.

Absence of a clinical correlation for complement-mediated, infection-enhancing antibodies in plasma or sera from HIV-1-infected individuals. Multicenter AIDS Cohort Study Group.

Neutralizing and complement-mediated infection-enhancing antibodies to HIV-1 were measured in sera or plasma from 54 HIV-1-positive individuals at various stages of disease, and from an additional 36 HIV-1-positive individuals for whom no clinical data were available. Antibodies were measured in microtiter infection assays utilizing MT-2 cells and the IIIB strain of HIV-1. The frequency of detection of both types of antibodies was identical, being 77 out of 90 cases (86%). Neutralizing and infection-enhancing antibodies were not always found together, and in four cases both were undetectable. No correlation was found between titers of either type of antibody and stage of disease. Furthermore, titers of infection-enhancing antibodies at early stages of disease did not predict rate of disease progression.

Contribution of sorbitol combined with activated charcoal in prevention of salicylate absorption.

The use of cathartics and activated charcoal in treating toxic ingestions has become a standard treatment modality. Sorbitol has been shown to be the most rapidly acting cathartic, but its therapeutic significance has been debated. Using a previously described aspirin overdose model, ten healthy volunteers participated in a crossover design study that investigated the effect of activated charcoal alone versus that of activated charcoal and sorbitol in preventing salicylate absorption. In phase 1 of the study, subjects consumed 2.5 g aspirin followed by 25 g activated charcoal one hour later. Urine was collected for 48 hours and analyzed for quantitative salicylate metabolites. Phase 2 was identical except that 1.5 g/kg sorbitol was consumed with the activated charcoal. The mean amount of aspirin absorbed without the use of sorbitol was 1.26 +/- 0.15 g, whereas the mean absorption was 0.912 +/- 0.18 g with the addition of sorbitol. This is a 28% decrease in absorption of salicylates attributable to the use of sorbitol. The difference is significant at P less than .05 by the paired Student's t test. This study demonstrates that the addition of sorbitol significantly decreases drug absorption in a simulated drug overdose model. Effects on absorption in actual overdose situations and on patient outcome should be the subjects of further study.

Rearrangement of enveloping layer cells without disruption of the epithelial permeability barrier as a factor in Fundulus epiboly.

Silver nitrate staining of blastoderms of Fundulus heteroclitus gastrulae shows that the number of marginal cells of the enveloping layer (EVL) is reduced from 160 to 25 during epiboly. To determine whether this decrease in the number of marginal cells was due to ingression, cell death, or rearrangement of cells, marginal and submarginal regions of the late gastrula were observed directly by time-lapse cinemicrography. Marginal cells rearrange to occupy submarginal positions by first narrowing their boundary with the external yolk syncytial layer (E-YSL), thus becoming tapered in shape. Then, the narrowed marginal boundary retracts from the E-YSL and moves submarginally in the plane of the epithelium. Concurrently, the marginal cells on both sides come into apposition; no gap or break appears in the circum-apical continuity of the epithelial sheet. Marginal cells leave the margin of the EVL during epiboly at a rate of about six per hour. The rate of movement of the EVL cells with respect to one another is about 0.5 to 1.0 micron/min at 21 degrees C. Submarginal cells rearrange in a similar fashion. Although no protrusive activity was seen at the lateral aspects of rearranging cells, the tapering or narrowing associated with rearrangement was accompanied by formation of microfolds on their apical surfaces, and separating or recently separated submarginal cells form "flowers" of microfolds on their apices adjacent to the site of separation. Morphometric analysis shows that about half the narrowing of the margin of the EVL during epiboly is accounted for by cell rearrangement and the other half by the associated tapering and narrowing. These results suggest that epiboly of the EVL may have an active component as well as a passive one.

The function and mechanism of convergent extension during gastrulation of Xenopus laevis.

The processes thought to function in Xenopus gastrulation include bottle cell formation, migration of cells on the roof of the blastocoel, and autonomous convergent extension of the circumblastoporal region. A review of recent and classical results shows that only the last accounts for the bulk of the tissue displacement of gastrulation, including spreading of the marginal zone toward the blastopore, involution of the marginal zone, and closure of the blastopore. Microsurgical manipulation and explantation studies, analysed by time-lapse video and cine microscopy, shows that the dorsal circumblastoporal region contains two regions which show either autonomous or semiautonomous convergent extension. The dorsal involuting marginal zone (IMZ) undergoes convergence (narrowing) and extension (lengthening) after its involution, beginning at the midgastrula stage and continuing through neurulation, such that it simultaneously extends posteriorly across the yolk plug and narrows the blastoporal circumference. Concurrently, the corresponding region of the overlying non-involuting marginal zone (NIMZ) begins a complementary convergent extension, but at a greater rate, which spreads vegetally to occupy surface area vacated by the IMZ. Tissue recombination experiments show that the deep cells of the dorsal IMZ bring about convergent extension. Labelling of small populations of these cells with a cell lineage tracer shows that convergent extension involves intercalation of deep cells to form a longer, narrower array. Direct time-lapse video and cine micrography of deep cells in cultured explants show that convergent extension involves radial and circumferential intercalation. Removal of the entire blastocoel roof of the early gastrula, including all or part of the NIMZ, shows that convergent extension of the IMZ alone can bring about its involution and blastopore closure. The role of convergent extension in gastrulation of other amphibians and other metazoans and its significance to related problems in early development are discussed.

Comparison of the effect of refrigerated versus room temperature media on the isolation of Neisseria gonorrhoeae from genital specimens.

We evaluated the effect of medium temperature at the time of inoculation on the isolation of Neisseria gonorrhoeae from urethral and cervical swabs. There were no major differences in the isolation rates of 176 positive cultures on cold or warm media. Colonies tended to be larger and more numerous on room temperature plates after 24 h; however, colonies on most refrigerated plates were easily recognized at 24 h, and growth was essentially the same on both plates after 48 h.

Neural crest cell behavior in white and dark larvae of Ambystoma mexicanum: time-lapse cinemicrographic analysis of pigment cell movement in vivo and in culture.

The pattern of migration and motile activity of developing pigment cells of the Mexican axolotl, Ambystoma mexicanum, were analyzed by time-lapse cinemicrography in vivo and in culture. In vivo, melanocytes of dark (D/-) larvae migrate from dorsal to ventral in a highly directional manner. They are elongated and aligned parallel to the direction of migration. Nearly all protrusive activity occurs at their ventral, leading edges. Translocation occurs at a mean rate of 0.7 micron/min and involves alternate or simultaneous advance of the leading and trailing edges of the cell. Indirect evidence suggests that cytoplasmic flow is common. Directional migration occurs in apparent absence of contact between melanocytes. In white (d/d) larvae, protrusive activity is infrequent and the melanocytes move slowly or not at all. Explanted neural crest cells of dark and white larvae attach, spread, and differentiate into melanophores and xanthophores in culture. Individual cultured cells are unbiased in direction of protrusive activity and path of migration. Centrifugal spreading occurs by contacting inhibition of movement. Distribution of protrusive activity, polarity, and contact behavior changes with developmental age in vivo and in culture in ways that may be important in establishing the pigment pattern.

Neural crest cell behavior in white and dark larvae of Ambystoma mexicanum: differences in cell morphology, arrangement, and extracellular matrix as related to migration.

Melanocytes of white (d/d) larvae of the Mexican axolotl (Ambystoma mexicanum) are confined to the dorsal midline of the trunk region, whereas in dark (D/-) larvae they are spread laterally on the flank as well, where they contribute to the normal pigment pattern of the trunk. Pigment cell migration in the subepidermal space of white larvae is inhibited by the white epidermis (Dalton '50; Keller et al., '82). The present scanning electron microscopic study describes a well-defined sequence of changes in shape and arrangement of neural crest cells during and after their segregation from the neural tube in both dark and white axolotls. The morphology of the neural crest cells migrating in the subepidermal pathway of dark larvae is correlated with their motile behavior and pattern of migration in vivo, as described by time-lapse cinemicrography (Keller and Spieth, '83). Also, the structures of the matrix material in the subepidermal space of dark and white axolotls differ in ways that may be related to the epidermal inhibition of migration in the latter. Numerous possibilities for contact guidance offered by the structure and topography of the substrata, neighboring cells, and the extracellular matrix in the migration path are described and discussed.

Perpendicular orientation and directional migration of amphibian neural crest cells in dc electrical fields.

The behavior of cultured neural crest cells of Ambystoma mexicanum and Xenopus laevis in dc electrical fields was studied. In fields of 1-5 V/cm, isolated or confluent cells retract both their anode- and cathode-facing margins. Subsequently, the cells elongate, with protrusive activity confined to their narrow ends. In larger fields (greater than or equal to 5 V/cm), protrusions form on the cathode-facing sides of the perpendicularly oriented cells. The cells then begin migrating laterally, perpendicular to their long axes, towards the cathode. We suggest that the perpendicular alignment and cathode-directed migrations result from cytoskeletal changes mediated by modified ion fluxes through the anode-facing (hyperpolarized) and cathode-facing (depolarized) cell membranes. The breaking of cellular confluence in response to dc electric fields is also discussed.

An experimental analysis of the role of bottle cells and the deep marginal zone in gastrulation of Xenopus laevis.

Earlier work suggested that the change in shape or the active migration of the bottle cells in the amphibian blastoporal region results in an invagination that comprises a major part of gastrulation. In the present study of gastrulation in Xenopus laevis, microsurgical extirpation and rearrangement experiments, analyzed with time-lapse cinemicrography and scanning electron microscopy, show that the bottle cells have a lesser role in gastrulation. Gastrulation is not a process of invagination but of involution of the deep and superficial layers of the marginal zone. Involution is dependent on unique properties of the cells in the deep marginal zone. In contrast, the superficial layer, including the bottle cells, does not have properties essential for involution but is passively moved inside to form the lining of the archenteron by what is probably an active migration of the underlying cells of the deep marginal zone. Bottle cells form by the shrinking and thickening of the superficial layer in the late blastula and early gastrula. They are moved inside, largely because of their attachment to the underlying deep cells, and then they spread and flatten in the latter half of gastrulation to form a large area of the lining of the periphery of the archenteron. The formation of the initial blastoporal groove by bending of the superficial cells sheet during bottle cell formation and the extension of the periphery of the archenteron during spreading of the bottle cells is the extent of the active contribution of bottle cells to the depth of the archenteron. The bulk of the depth is generated by the vegetal extension of the marginal zone and the movement of the involuted deep cells toward the animal pole.

The cellular basis of epiboly: an SEM study of deep-cell rearrangement during gastrulation in Xenopus laevis.

Measurements of several indices of shape, contact, position and arrangement of deep cells in the late blastula and gastrula were made from scanning electron micrographs of carefully staged, fractured embryos in order to describe the cellular processes which account for the increased area of the deep region of the gastrula during extension of the dorsal marginal zone and epiboly of the animal region. At the onset of gastrulation, the deep cells of the dorsal marginal zone become elongated, extend protrusions between one another along radii of the embryo and interdigitate to form fewer layers of cells of greater area in a process of radial interdigitation. When interdigitation, is complete, the deep region consists of one layer of columnar cells which then flatten and spread and thus account for additional increase in area of the deep region. During epiboly of the animal region, interdigitation occurs and the number of cell layers decreases without the changes in cell shape seen in the dorsal marginal zone. These differences may be related to the anisotropy of expansion (extension and convergence) in the dorsal marginal done as opposed to uniform spreading in the animals region, or they may reflect an active cell motility in the dorsal marginal zone as opposed to a passive behavior in the animal region. A cellular and mechanical model is presented in which active (autonomous) spreading is brought about by active, force-producing interdigitation and subsequent flattening of deep cells. A model of passive spreading (stretching) is also presented. These observations suggest experiments that would determine the relationship of cell behavior to the mechanics of gastrulation.