Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

Monte Carlo modeling of polarized light propagation: Stokes vs Jones - Part I

This bipartite comparative study aims at inspecting the similarities and differences between the Jones and Stokes–Mueller formalisms when modeling polarized light propagation with numerical simulations of the Monte Carlo type. In this first part, we review the theoretical concepts that concern light propagation and detection with both pure and partially/totally unpolarized states. The latter case involving fluctuations, or “depolarizing effects,” is of special interest here: Jones and Stokes–Mueller are equally apt to model such effects and are expected to yield identical results. In a second, ensuing paper, empirical evidence is provided by means of numerical experiments, using both formalisms.

Monte Carlo modeling of polarized light propagation: Stokes vs Jones - Part II

In this second part of our comparative study inspecting the (dis)similarities between “Stokes” and “Jones,” we present simulation results yielded by two independent Monte Carlo programs: (i) one developed in Bern with the Jones formalism and (ii) the other implemented in Ulm with the Stokes notation. The simulated polarimetric experiments involve suspensions of polystyrene spheres with varying size. Reflection and refraction at the sample/air interfaces are also considered. Both programs yield identical results when propagating pure polarization states, yet, with unpolarized illumination, second order statistical differences appear, thereby highlighting the pre-averaged nature of the Stokes parameters. This study serves as a validation for both programs and clarifies the misleading belief according to which “Jones cannot treat depolarizing effects.”

Delegation Proxies: The Power of Propagation

Scoping behavioral variations to dynamic extents is useful to support non-functional requirements that otherwise result in cross-cutting code. Unfortunately, such variations are difficult to achieve with traditional reflection or aspects. We show that with a modification of dynamic proxies, called delegation proxies, it becomes possible to reflectively implement variations that propagate to all objects accessed in the dynamic extent of a message send. We demonstrate our approach with examples of variations scoped to dynamic extents that help simplify code related to safety, reliability, and monitoring.

Propagation of melting cooperativity along the phosphodiester backbone of DNA

The role of the DNA phosphodiester backbone in the transfer of melting cooperativity between two helical domains was experimentally addressed with a helix-bulge-helix DNA model, in which the bulge consisted of a varying number of either conformationally flexible propanediol or conformationally constrained bicyclic anucleosidic phosphodiester backbone units. We found that structural communication between two double helical domains is transferred along the DNA backbone over the equivalent of ca. 12-20 backbone units, depending on whether there is a symmetric or asymmetric distribution of the anucleosidic units on both strands. We observed that extension of anucleosidic units on one strand only suffices to disrupt cooperativity transfer in a similar way as if extension occurs on both strands, indicating that the length of the longest anucleosidic inset determines cooperativity transfer. Furthermore, conformational rigidity of the sugar unit increases the distance of coopertivity transfer along the phosphodiester backbone. This is especially the case when the units are asymmetrically distributed in both strands

Auxin Regulates the Initiation and Radial Position of Plant Lateral Organs

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical–basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical–basal position or the identity of the induced structures.

Burrowing in Marine Muds by Crack Propagation: Kinematics and Forces

The polychaete Nereis virens burrows through muddy sediments by exerting dorsoventral forces against the walls of its tongue-depressor- shaped burrow to extend an oblate hemispheroidal crack. Stress is concentrated at the crack tip, which extends when the stress intensity factor (K-I) exceeds the critical stress intensity factor (K-Ic). Relevant forces were measured in gelatin, an analog for elastic muds, by photoelastic stress analysis, and were 0.015 +/- 0.001 N (mean +/- s.d.;N= 5). Measured elastic moduli (E) for gelatin and sediment were used in finite element models to convert the forces in gelatin to those required in muds to maintain the same body shapes observed in gelatin. The force increases directly with increasing sediment stiffness, and is 0.16 N for measured sediment stiffness of E=2.7x10(4) Pa. This measurement of forces exerted by burrowers is the first that explicitly considers the mechanical behavior of the sediment. Calculated stress intensity factors fall within the range of critical values for gelatin and exceed those for sediment, showing that crack propagation is a mechanically feasible mechanism of burrowing. The pharynx extends anteriorly as it everts, extending the crack tip only as far as the anterior of the worm, consistent with wedge-driven fracture and drawing obvious parallels between soft-bodied burrowers and more rigid, wedge-shaped burrowers(i.e. clams). Our results raise questions about the reputed high energetic cost of burrowing and emphasize the need for better understanding of sediment mechanics to quantify external energy expenditure during burrowing.

Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

In vitro propagation of Glandularia peruviana (L.) Small, an ornamental native plant from South America

The flower market is characterized by being both eager for novelties and highly competitive. The exploration of native species with ornamental potential represents a remarkable area of research, since it entails the introduction and development of novel promising ornamental crops. The genus Glandularia, widely distributed in Argentina, holds an enormous ornamental potential, due to the variety of colors of its inflorescences (red, violet, white, rose and lily), and extended flowering period. There is little information on tissue culture of Glandularia, thus highlighting the relevance of this research. In this work, the conditions for in vitro multiplication of G. peruviana were optimized. It was concluded that WPM supplemented with TDZ, in concentrations ranging from 1.1 to 9.0 μM, was the most adequate treatment, rendering a multiplication rate of approximately 10 de novo shoots per explant. This paper presents a protocol for the in vitro propagation of this species and introduces interesting prospects in the application of biotechnological tools to breed Glandularia.

Observations on the propagation of internal tidal waves on the continental slope off Sierra Leone

During R/V Meteor-cruise no. 30 4 moorings with 17 current meters were placed on the continental slope of Sierra Leone at depths between 81 and 1058 meters. The observation period started on March 8, 1973, 16.55 hours GMT and lasted 19 days for moorings M30_068MOOR, M30_069MOOR, M30_070MOOR on the slope and 9 days for M30_067MOOR on the shelf. One current meter recorded at location M30_067MOOR for 22 days. Hydrographic data were collected at 32 stations by means of the "Kieler Multi-Meeressonde". Harmonic analysis is applied to the first 15 days of the time series to determine the M2 and S2 tides. By vertically averaging of the Fourier coefficients the field of motion is separated into its barotropic and its baroclinic component. The expected error generated by white Gaussian noise is estimated. To estimate the influence of the particular vertical distribution of the current meters, the barotropic M2 tide is calculated by ommitting and interchanging time series of different moorings. It is shown that only the data of moorings M30_069MOOR, M30_070MOOR and M30_067MOOR can be used. The results for the barotropic M2 tide agree well with the previous publications of other authors. On the slope at a depth of 1000 m there is a free barotropic wave under the influence of the Coriolis-force propagating along the slope with an amplitude of 3.4 cm S**-1. On the shelf, the maximum current is substantially greater (5.8 cm s**-1) and the direction of propagation is perpendicular to the slope. As for the continental slope a separation into different baroclinic modes using vertical eigenmodes is not reasonable, an interpretation of the total baroclinic wave field is tried by means of the method of characteristis. Assuming the continental slope to generate several linear waves, which superpose, baroclinic tidal ellipses are calculated. The scattering of the direction of the major axes M30_069MOOR is in contrast to M30_070MOOR, where they are bundled within an angle of 60°. This is presumably caused by the different character of the bottom topography in the vicinity of the two moorings. A detailed discussion of M30_069MOOR is renounced since the accuracy of the bathymetric chart is not sufficient to prove any relation between waves and topography. The bundeling of the major axes at M30_070MOOR can be explained by the longslope changes of the slope, which cause an energy transfer from the longslope barotropic component to the downslope baroclinic component. The maximum amplitude is found at a depth of 245 m where it is expected from the characteristics originating at the shelf edge. Because of the dominating barotropic tide high coherence is found between most of the current meters. To show the influence of the baroclinic tidal waves, the effect of the mean current is considered. There are two periods nearly opposite longshore mean current. For 128 hours during each of these periods, starting on March 11, 05.00, and March 21, 08.30, the coherences and energy spectra are calculated. The changes in the slope of the characteristics are found in agreement with the changes of energy and coherence. Because of the short periods of nearly constant mean current, some of the calculated differences of energy and coherence are not statistically significant. For the M2 tide a calculation of the ratios of vertically integrated total baroclinic energy and vertically integrated barotropic kinetic energy is carried out. Taking into account both components (along and perpendicular to the slope) the obtained values are 0.75 and 0.98 at the slope and 0.38 at the shelf. If each component is considered separately, the ratios are 0.39 and 1.16 parallel to the slope and 5.1 and 15.85 for the component perpendicular to it. Taking the energy transfer from the longslope component to the doenslope component into account, a simple model yields an energy-ratio of 2.6. Considering the limited application of the theory to the real conditions, the obtained are in agreement with the values calculated by Sandstroem.

Elevated pCO2 from late summer coastal upwellings in the western Baltic Sea enhances vegetative growth in the brown algae Fucus serratus: a laboratory assessment

The brown algae Fucus serratus is one of the major meadow forming algae of the Western Baltic Sea nearshore ecosystem. At the end of summer, those meadows are exposed to local upwelling suddenly increasing the pCO2 and DIC up to 2500 µatm and 2250 µmol/kg resp., for period of days to weeks. This study investigates the growth response of summer's vegetative Fucus serratus to elevated pCO2 (1350 and 4080 µatm) during a 40 days laboratory incubation. After 10 days, increases of growth rates of 20 % and 47 % of the control were observed in the 1350 and 4080 µatm pCO2 treatments respectively. Beyond 20 days, the growth rates collapsed in all treatments due to nutrients shortage, as demonstrated by high C:N ratios (95:1) and low N tissue content (0.04 % of dry weight). The collapse occurs faster at higher pCO2. On day 30, growth rates were reduced by 40 % and 100 % relative to the control at 1350 and 4080 µatm respectively. These results are consistent with a fertilizing effect of elevated pCO2 on Fucus serratus presumably linked to the transition from active HCO3- to passive CO2(aq) uptake. This positive effect is limited by nutrients resources, low seawater dissolved inorganic N and P and shortage of the nutrients reserves accumulated over the previous autumn and winter.