Establishment and characterization of a primary and a metastatic melanoma cell line from Grey horses

The Grey horse phenotype, caused by a 4.6 kb duplication in Syntaxin 17, is strongly associated with high incidence of melanoma. In contrast to most human melanomas with an early onset of metastasis, the Grey horse melanomas have an extended period of benign growth, after which 50% or more eventually undergo progression and may metastasize. In efforts to define changes occurring during Grey horse melanoma progression, we established an in vitro model comprised of two cell lines, HoMel-L1 and HoMel-A1, representing a primary and a metastatic stage of the melanoma, respectively. The cell lines were examined for their growth and morphological characteristics, in vitro and in vivo oncogenic potential, chromosome numbers, and expression of melanocytic antigens and tumor suppressors. Both cell lines exhibited malignant characteristics; however, the metastatic HoMel-A1 showed a more aggressive phenotype characterized by higher proliferation rates, invasiveness, and a stronger tumorigenic potential both in vitro and in vivo. HoMel-A1 displayed a near-haploid karyotype, whereas HoMel-L1 was near-diploid. The cell lines expressed melanocytic lineage markers such as TYR, TRP1, MITF, PMEL, ASIP, MC1R, POMC, and KIT. The tumor suppressor p53 was strongly expressed in both cell lines, while the tumor suppressors p16 and PTEN were absent in HoMel-A1, potentially implicating significance of these pathways in the melanoma progression. This in vitro model system will not only aid in understanding of the Grey horse melanoma pathogenesis, but also in unraveling the steps during melanoma progression in general as well as being an invaluable tool for development of new therapeutic strategies.     

Plasticity of rhizosphere hydraulic properties as a key for efficient utilization of scarce resources

Background

It is known that the soil near roots, the so-called rhizosphere, has physical and chemical properties different from those of the bulk soil. Rhizosphere properties are the result of several processes: root and soil shrinking/swelling during drying/wetting cycles, soil compaction by root growth, mucilage exuded by root caps, interaction of mucilage with soil particles, mucilage shrinking/swelling and mucilage biodegradation. These processes may lead to variable rhizosphere properties, i.e. the presence of air-filled gaps between soil and roots; water repellence in the rhizosphere caused by drying of mucilage around the soil particles; or water accumulation in the rhizosphere due to the high water-holding capacity of mucilage. The resulting properties are not constant in time but they change as a function of soil condition, root growth rate and mucilage age.

Scope

We consider such a variability as an expression of rhizosphere plasticity, which may be a strategy for plants to control which part of the root system will have a facilitated access to water and which roots will be disconnected from the soil, for instance by air-filled gaps or by rhizosphere hydrophobicity. To describe such a dualism, we suggest classifying rhizosphere into two categories: class A refers to a rhizosphere covered with hydrated mucilage that optimally connects roots to soil and facilitates water uptake from dry soils. Class B refers to the case of air-filled gaps and/or hydrophobic rhizosphere, which isolate roots from the soil and may limit water uptake from the soil as well water loss to the soil. The main function of roots covered by class B will be long-distance transport of water.

Outlook

This concept has implications for soil and plant water relations at the plant scale. Root water uptake in dry conditions is expected to shift to regions covered with rhizosphere class A. On the other hand, hydraulic lift may be limited in regions covered with rhizosphere class B. New experimental methods need to be developed and applied to different plant species and soil types, in order to understand whether such dualism in rhizosphere properties is an important mechanism for efficient utilization of scarce resources and drought tolerance.     

Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34⁺ hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2–3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.     

Challenges in imaging and predictive modeling of rhizosphere processes

Background

Plant-soil interaction is central to human food production and ecosystem function. Thus, it is essential to not only understand, but also to develop predictive mathematical models which can be used to assess how climate and soil management practices will affect these interactions.

Scope

In this paper we review the current developments in structural and chemical imaging of rhizosphere processes within the context of multiscale mathematical image based modeling. We outline areas that need more research and areas which would benefit from more detailed understanding.

Conclusions

We conclude that the combination of structural and chemical imaging with modeling is an incredibly powerful tool which is fundamental for understanding how plant roots interact with soil. We emphasize the need for more researchers to be attracted to this area that is so fertile for future discoveries. Finally, model building must go hand in hand with experiments. In particular, there is a real need to integrate rhizosphere structural and chemical imaging with modeling for better understanding of the rhizosphere processes leading to models which explicitly account for pore scale processes.

     

Root age distribution: how does it matter in plant processes? A focus on water uptake

Aims and background

Root growth creates a gradient in age at both the scale of the single root, from distal to proximal parts, but also at the root system level when young branch roots emerge from the axis or new nodal roots are emitted that may reach same soil domain as older roots. It is known that a number of root functions will vary with root type and root tissue age (e.g. respiration, exudation, ion uptake, root hydraulic conductance, mucilage release…) and so will the resulting rhizosphere properties. The impact of the distribution of root demography with depth, and related functions, on the overall functioning of the root system is fundamental for an integration of processes at the root system scale.

Scope and conclusion

Starting from methods for measuring root demography, we discuss the availability of data related to root age and its spatial distribution, considering plant types (monocot/dicot, perennial/annuals) which may exhibit different patterns. We then give a detailed review of variation of root/rhizosphere properties related to root age, focusing on root water uptake processes. We examine the type of response of certain properties to changes in age and whether a functional relationship can be derived. Integration of changing root properties with age into modelling approaches is shown from 3D models at the single plant scale to approaches at the field scale based on integrated root system age. Functional structural modelling combined with new development in non-invasive imaging of roots show promises for integrating influence of age on root properties, from the local to whole root system scales. However, experimental quantification of these properties, such as hydraulic conductance variation with root age and root types, or impact of mucilage and its degradation products on rhizosphere hydraulic properties, presently lag behind the theoretical developments and increase in computational power.

     

Rhizosphere: biophysics, biogeochemistry and ecological relevance

Life on Earth is sustained by a small volume of soil surrounding roots, called the rhizosphere. The soil is where most of the biodiversity on Earth exists, and the rhizosphere probably represents the most dynamic habitat on Earth; and certainly is the most important zone in terms of defining the quality and quantity of the Human terrestrial food resource. Despite its central importance to all life, we know very little about rhizosphere functioning, and have an extraordinary ignorance about how best we can manipulate it to our advantage. A major issue in research on rhizosphere processes is the intimate connection between the biology, physics and chemistry of the system which exhibits astonishing spatial and temporal heterogeneities. This review considers the unique biophysical and biogeochemical properties of the rhizosphere and draws some connections between them. Particular emphasis is put on how underlying processes affect rhizosphere ecology, to generate highly heterogeneous microenvironments. Rhizosphere ecology is driven by a combination of the physical architecture of the soil matrix, coupled with the spatial and temporal distribution of rhizodeposits, protons, gases, and the role of roots as sinks for water and nutrients. Consequences for plant growth and whole-system ecology are considered. The first sections address the physical architecture and soil strength of the rhizosphere, drawing their relationship with key functions such as the movement and storage of elements and water as well as the ability of roots to explore the soil and the definition of diverse habitats for soil microorganisms. The distribution of water and its accessibility in the rhizosphere is considered in detail, with a special emphasis on spatial and temporal dynamics and heterogeneities. The physical architecture and water content play a key role in determining the biogeochemical ambience of the rhizosphere, via their effect on partial pressures of O₂ and CO₂, and thereby on redox potential and pH of the rhizosphere, respectively. We address the various mechanisms by which roots and associated microorganisms alter these major drivers of soil biogeochemistry. Finally, we consider the distribution of nutrients, their accessibility in the rhizosphere, and their functional relevance for plant and microbial ecology. Gradients of nutrients in the rhizosphere, and their spatial patterns or temporal dynamics are discussed in the light of current knowledge of rhizosphere biophysics and biogeochemistry. Priorities for future research are identified as well as new methodological developments which might help to advance a comprehensive understanding of the co-occurring processes in the rhizosphere.     

Use of a microtensiometer technique to study hydraulic lift in a sandy soil planted with pearl millet (Pennisetum americanum [L.] Leeke)

A pot experiment was carried out with pearl millet (Pennisetum americanum [L.] Leeke) growing in a sandy soil in which the upper (topsoil) and lower (subsoil) parts of the pots were separated by a perlite layer to prevent capillary water movement. Using microtensiometers a study was made to establish whether it was possible to measure hydraulic lift by which the upper part of the soil was rewetted when water was supplied exclusively to the lower part of the soil.Hydraulic lift occurred during the first seven days of the period of measurement, with a maximum water release to the soil of 2.7 Vol. % during one night (equivalent to 10.8 mL water in the top 10 cm of the soil profile). This magnitude was obtained at very high root length densities, so that water release from the roots would be expected to be much smaller under field conditions.Hydraulic lift ceased when the soil matric potential in the topsoil dropped below-10 kPa at the end of the light period and could not be re-established, neither by extending the dark period, nor after rewatering the topsoil. The disappearance of hydraulic lift could be explained in part through osmotic adaptation of plant roots and, thus prevention of water release from the roots in the topsoil. It is concluded that hydraulic lift may affect nutrient uptake from drying topsoil by extending the time period favourable for uptake from the topsoil.     

Phosphorus availability in different types of open-cast mine spoil and the potential impact of organic matter application

Greenhouse experiments were conducted in order to determine for carboniferous and non-carboniferous mine spoil substrates from the Lusatian lignite mining area (i) the suitable extraction method for plant available P, (ii) the soil capacity for immobilisation of P and (iii) the impact of sewage sludge and compost on P availability. Ca-lactate extraction (DL) and NH₄F-extraction (Bray) were both suited equally well for the determination of plant available P as they extracted similar amounts of P on both spoils, they showed a close correlation with each other (R=0.97 ²) and they showed a close relation with plant P uptake (R²=0.63 and R²=0.66, respectively). Phosphorus recovery from limed carboniferous mine spoil five days after mineral fertiliser application was only 50%, and decreased to 30% after 54 days. As pH was increased from 3.0 to 5.0 the amount of P immobilised decreased only by about 5%. Several pH dependent processes of P immobilisation and release could occur concurrently counteracting each other. One process could be P sorption to newly formed hydroxy-Al-surfaces but P desorption could also take place as pH increases by decreasing surface positive charge. Finally, due to high Ca concentrations in spoil solution formation of Ca-phosphates, even at lower pH values, cannot be excluded as a possible mechanism of P immobilisation. As part of the P is bound in organic matter, application of P with organic matter resulted in a lower P recovery compared to mineral P-fertiliser. However, the amount of P recovered did not differ between carboniferous and non-carboniferous mine spoil, if P was applied in the form of organic matter, indicating that the application of P with organic matter might be a measure to overcome P immobilisation in carboniferous mine spoils. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion

In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.