Permeable collagen-glycosaminoglycan cross-linked copolymers for the study of biological responses of coculturede Sertoli and spermatogenic cells

A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on nonpermeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15–60 min) and dose-dependent (0.1–10µM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.Abbreviations C-GAG, collagen type I-glycosaminoglycans - C-C6S, collagen type I-chondroitin-6-sulfate - C-H, collagen type I-heparin     

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications.     

Nutrient transfer between the root zones of soybean and maize plants connected by a common mycorrhizal mycelium

The objective of the study was to determine whether nutrient fluxes mediated by hyphae of vesicular-arbuscular mycorrhizal (VAM) fungi between the root zones of grass and legume plants differ with the legume's mode of N nutrition. The plants, nodulating or nonnodulating isolines of soybean [ Glycine max (L.) Merr.], were grown in association with a dwarf maize ( Zea mays L.) cultivar in containers which interposed a 6-cm-wide root-free soil bridge between legume and grass container compartments. The bridge was delimited by screens (44 μm) which permitted the passage of hyphae, but not of roots and minimized non VAM interactions between the plants. All plants were colonized by the VAM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe. The effects of N input to N-sufficient soybean plants through N₂-fixation or N-fertilization on associated maize-plant growth and nutrition were compared to those of an N-deficient (nonnodulating, unfertilized) soybean control. Maize, when associated with the N-fertilized soybean, increased 19% in biomass, 67% in N content and 77% in leaf N concentration relative to the maize plants of the N-deficient association. When maize was grown with nodulated soybean, maize N content increased by 22%, biomass did not change, but P content declined by 16%. Spore production by the VAM fungus was greatest in the soils of both plants of the N-fertilized treatment. The patterns of N and P distribution, as well as those of the other essential elements, indicated that association with the N-fertilized soybean plants was more advantageous to maize than was association with the N₂-fixing ones.     

Silicon accumulation and water uptake by wheat

Silicon (Si) content in cereal plants and soil-Si solubility may be used to estimate transpiration, assuming passive Si uptake. The hypothesis for passive-Si uptake by the transpiration stream was tested in wheat (Triticum aestivum cv. Stephens) grown on the irrigated Portneuf silt loam soil (Durixerollic calciorthid) near Twin Falls, Idaho. Treatments consisted of 5 levels of plant-available soil water ranging from 244 to 776 mm provided primarily by a line-source sprinkler irrigation system. Evapotranspiration was determined by the water-balance method and water uptake was calculated from evapotranspiration, shading, and duration of wet-surface soil. Water extraction occurred from the 0 to 150-cm zone in which equilibrium Si solubility (20°C) was 15 mg Si L^–1 in the A_p and B_k (0–58 cm depth) and 23 mg Si L^–1 in the B_kq (58–165 cm depth).At plant maturity, total Si uptake ranged from 10 to 32 g m^–2, above-ground dry matter from 1200 to 2100 g m^–2 and transpiration from 227 to 546 kg m^–2. Silicon uptake was correlated with transpiration (Si_up=–07+06T, r²=0.85) and dry matter yield with evapotranspiration (Y=119+303ET, r²=0.96). Actual Si uptake was 2.4 to 4.7 times that accounted for by passive uptake, supporting designation of wheat as a Si accumulator. The ratio of Si uptake to water uptake increased with soil moisture. The confirmation of active Si uptake precludes using Si uptake to estimate water use by wheat.     

Associative N2-fixation in plants growing in saline sodic soils and its relative quantification based on15N natural abundance

The interactive effect of low P supply (0, 10, 20 and 40 M) and plant age on nodule number, mass and functioning (ureide analysis technique), vegetative growth and pod production were investigated in glasshouse-grown nodulated cowpea (Vigna unguiculata L.cv. Kausband) in sand culture. Compared with 40 M P, P stress (0 M P) or very low (10 M P) supply markedly impaired nodulation, allantoin and amino-N concentrations and weight of N solutes in xylem exudates. Consequently, P stress reduced top growth and pod yields by 48 and 90%, respectively. N solutes in xylem exudates and total plant N assayed by Kjeldahl technique (as estimates of N₂ fixation) responded similarly to P supply. However, the relative ureide index [(ureide-N/ureide N+amino-N)×100] remained constant (99%), irrespective of P supply, indicating the plants' complete dependency on symbiosis for growth, without implying that growth was markedly increased by N₂ fixation. Although P concentrations in plant tops, roots and nodules increased with P supply, N concentrations in these plant tissues were unaffected by P supply. The concentrations of N and P in the nodules were 2–2 1/2 times higher than in plant tops. P application interacted strongly with plant age, with the largest P effect evidently achieved at the early podding stage. The significance and implications of these results are discussed.     

Mechanisms of iron acquisition from siderophores by microorganisms and plants

Most bacteria, fungi, and some plants respond to Fe stress by the induction of high-affinity Fe transport systems that utilize biosyrthetic chelates called siderophores. To competitively acquire Fe, some microbes have transport systems that enable them to use other siderophore types in addition to their own. Bacteria such as Escherichia coli achieve this ability by using a combination of separate siderophore receptors and transporters, whereas other microbial species, such as Streptomyces pilosus, use a low specificity, high-affinity transport system that recognizes more than one siderophore type. By either strategy, such versatility may provide an advantage under Fe-limiting conditions; allowing use of siderophores produced at another organism's expense, or Fe acquisition from siderophores that could otherwise sequester Fe in an unavailable form.Plants that use microbial siderophores may also be more Fe efficient by virtue of their ability to use a variety of Fe sources under different soil conditions. Results of our research examining Fe transport by oat indicate parity in plant and microbial requirements for Fe and suggest that siderophores produced by root-colonizing microbes may provide Fe to plants that can use the predominant siderophore types. In conjunction with transport mechanisms, ecological and soil chemical factors can influence the efficacy of siderophores and phytosiderophores. A model presented here attempts to incorporate these factors to predict conditions that may govern competition for Fe in the plant rhizosphere. Possibly such competition has been a factor in the evolution of broad transport capabilities for different siderophores by microorganisms and plants.     

Calcium (45Ca) accumulation and transport in chicory (Cichorium intybus L.) root during bud development (forcing)

The lower part (4 cm) of the witloof chicory tap-root (15 cm) was immersed in a complete nutrient solution for 21 days, in the darkness at 18°C and at high RH. This process of forcing which leads to the emergence of an etiolated bud (chicon) was associated with a decrease in root dry weight. Although the amount of calcium in the root and the root cationic exchange capacity remained constant during forcing, the net uptake of calcium, negligible at the onset of forcing, progressively increased to a rate after ten days of 45 mol day^–1. Absorption of ⁴⁵Ca remained at a constant high rate, while the initially low upward migration of ⁴⁵Ca within the root and the chicon accelerated markedly. This upward migration was associated with a progressive decline in the release of newly absorbed ⁴⁵Ca. The data support the hypothesis that calcium acquisition by witloof chicory root is predominantly determined by calcium efflux. As the forcing progressed, the influx remained almost constant while a large decrease in the efflux led to a net uptake of calcium. Upward translocation was probably linked to the formation of new negative exchange sites within the growing chicon. The hypothesis that calcium movement occurred along a preferential pathway (xylem vessels) or involved a mass movement through the root is discussed.     

Mass-spectrometric determination of O2 and CO2 gas exchange in illuminated higher-plant cells

In order to estimate photosynthetic and respiratory rates in illuminated photoautotrophic cells of carnation (Dianthus caryophyllus L.), simultaneous measurements of CO₂ and O₂ gas exchange were performed using ¹⁸O₂, ¹³CO₂ and a mass-spectrometry technique. This method allowed the determination, and thus the comparison, of unidirectional fluxes of O₂ and CO₂. In optimum photosynthetic conditions (i.e. in the presence of high light and a saturating level of CO₂), the rate of CO₂ influx represented 75±5% of the rate of gross O₂ evolution. After a dark-to-light transition, the rate of CO₂ efflux was inhibited by 50% whereas the O₂-uptake rate was little affected. The effect of a recycling of respiratory CO₂ through photosynthesis on the exchange of CO₂ gas was investigated using a mathematical model. The confliction of the experimental data with the simulated gas-exchange rates strongly supported the view that CO₂ recycling was a minor event in these cells and could not be responsible for the observed inhibition of CO₂ efflux. On the basis of this assumption it was concluded that illumination of carnation cells resulted in a decrease of substrate decarboxylations, and that CO₂ efflux and O₂ uptake were not as tightly coupled in the light as in the dark. Furthermore, it could be calculated from the rate of gross photosynthesis that the chloroplastic electron-transport chain produced enough ATP in the light to account for the measured CO₂-uptake rate without involving cyclic transfer of electrons around PS I or mitochondrial supplementation.Abbreviations Chl chlorophyll - K_d permeability coefficient The authors thank Drs A. Vermeglio and P. Thibault, Dépt. de Biologie, CEN-Cadarache, St. Paul Lez Durance, France, for helpful discussions.     

Fine structure of plasmodesmata in mature leaves of sugarcane

The fine structure of plasmodesmata in vascular bundles and contiguous tissues of mature leaf blades of sugarcane (Saccharum interspecific hybrid L62–96) was studied with the transmission electron microscope. Tissues were fixed in glutaraldehyde, with and without the addition of tannic acid, and postfixed in OsO₄. The results indicate that the fine structure of plasmodesmata in sugarcane differs among various cell combinations in a cell-specific manner, but that three basic structural variations can be recognized among plasmodesmata in the mature leaf: 1) Plasmodesmata between mesophyll cells. These plasmodesmata possess amorphous, electron-opaque structures, termed sphincters, that extend from plasma membrane to desmotubule near the orifices of the plasmodesmata. The cytoplasmic sleeve is filled by the sphincters where they occur; elsewhere it is open and entirely free of particulate or spokelike components. The desmotubule is tightly constricted and has no lumen within the sphincters, but between the sphincters it is a convoluted tubule with an open lumen. 2) Plasmodesmata that traverse the walls of chlorenchymatous bundle-sheath cells and mestome-sheath cells. In addition to the presence of sphincters, these plasmodesmata are modified by the presence of suberin lamellae in the walls. Although the plasmodesmata are quite narrow and the lumens of the desmotubules are constricted where they traverse the suberin lamellae, the cytoplasmic sleeves are still discernible and appear to contain substructural components there. 3) Plasmodesmata between parenchymatous cells of the vascular bundles. These plasmodesmata strongly resemble those found in the roots of Azolla, in that their desmotubules are closed for their entire length and their cytoplasmic sleeves appear to contain substructural components for their entire length. The structural variations exhibited by the plasmodesmata of the sugarcane leaf are compared with those proposed for a widely-adopted model of plasmodesmatal structure.Abbreviation ER endoplasmic reticulum This study was supported by National Science Foundation grants DCB 87-01116 and DCB 90-01759 to R.F.E. and a University of Wisconsin-Madison Dean's Fellowship to K. R.-B. We also thank Claudia Lipke and Kandis Elliot for photographic and artistic assistance, respectively.