A free-radical hypothesis for the instability and evolution of genotype and phenotypein vitro

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA     

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.     

Effect of phosphorus supply to the surface roots of wheat on root extension and rhizosphere chemistry in an acidic subsoil

Seedlings of two cultivars of wheat (Triticum aestivum L.) differing in tolerance to aluminium (Al) were grown using a split-root sand/soil culture technique. Each culture tube was divided horizontally into a surface (0–150 mm) compartment and a subsurface (150–250 mm) compartment separated by a root-permeable paraffin wax barrier. Thus phosphorus (P) supplied to surface roots could not percolate or diffuse into the soil in the subsurface compartment. The soil in the subsurface compartment was divided into ‘rhizosphere’ and ‘non-rhizosphere’ zones using a porous (5 μm) membrane. Root growth of both cultivars into the subsurface zone was enhanced by increased P supply to surface roots, but did not conform to known relationships between root growth and soil pH, extractable-Al, or pH, Al or P concentrations in soil solution. Concentrations of Al in soil solution in the rhizosphere were greater than those in solution in the bulk soil. Concentrations of Al reactive with pyrocatechol violet (30s-RRAI) in the rhizosphere soil solution were generally greater than those in non-rhizosphere soil. With the Al-sensitive cultivar, root dry weight and length increased as concentrations of RRAl in the rhizosphere soil solution increased. Increased concentrations of Al in rhizosphere soil solutions were not related to the presence of organic ligands in solution. The effect of P in promoting root penetration into the acidic subsurface stratum was not related to differential attainment of maturity by the plant shoots, but appeared to be related to the effect of P in enhancing the rate of root growth. Thus, suboptimal supply of P to the surface roots of a plant, even at levels sufficient to preclude development of nutritional (P) stress symptoms, may seriously reduce tolerance to Al, and hence diminish the ability of roots to penetrate into acidic subsoils.     

An inflatable minirhizotron system for root observations with improved soil/tube contact

Commonly used minirhizotrons consisting of a transparent tube inserted into the soil seldom attain good contact between the tube and the soil, which leads to root growth occurring in a gap rather than in the soil. A new system is described involving an inflatable flexible rubber wall, made from a modified motorcycle tube. Pressure ensures a proper tube/soil contact so that the environmental circumstances for root growth along the tube more closely correspond to those in the undisturbed soil. Before the endoscope slide is introduced into the minirhizotron for taking pictures, the inflatable tube is removed, so that there is no-often opaque-wall between the endoscope and the roots. This improves the picture quality and facilitates the analysis of root images.     

Phosphorus efficiency of plants

Föhse et al. (1988) have shown that P influx per unit root length in seven plant species growing in a low-P soil varied from 0.6×10^-14 to 4.8×10^-14 mol cm^-1s^-1. The objective of this work was to investigate the reasons for these differences. No correlation was found between P influx and root radius, root hairs, cation-anion balance and Ca uptake. However, when root hairs were included in mathematical model calculations, the differences of P influx could be accounted for. These calculations have shown that in soils low in available P, contribution to P uptake by root hairs was up to 90% of total uptake. The large contribution of root hairs to P uptake was partly due to their surface area, which was similar to that of the root cylinder. However, the main reason for the high P uptake efficiency of root hairs was their small radius (approx. 5×10^-4 cm) and their perpendicular growth into the soil from the root axis. Because of the small radius compared to root axes, P concentration at root hair surfaces decreased at a slower pace and therefore P influx remained higher. Under these conditions higher I_max (maximum influx) or smaller K_m values (Michaelis constant) increased P influx. The main reasons for differences found in P influx among species were the size of I_max and the number and length of root hairs. In a soil low in available P, plant species having more root hairs were able to satisfy a higher proportion of their P demand required for maximum growth.     

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.     

Kinetic and molecular orbital studies on the rate of oxidation of monosubstituted phenols and anilines by lactoperoxidase compound II in comparison with the case of horseradish peroxidase

The second-order rate constant (k4) for the oxidation of monosubstituted phenols and anilines by lactoperoxidase compound II was examined by Chance's method [B. Chance, Arch. Biochem. Biophys. 71 (1957), 130–136]. When the electronic states of these substrates were calculated by an ab initio molecular orbital method, it was found that the log k4 value correlates well with the highest occupied molecular orbital (HOMO) energy level but not with the net charge or frontier electron density. These results are essentially similar to those reported previously in the case of horseradish peroxidase [J. Sakurada, R. Sekiguchi, K. Sato, and T. Hosoya, Biochemistry 29 (1990), 4093–4098], showing some dissimilar features which are considered to reflect the structural difference between the two enzymes.Abbreviations HOMO highest occupied molecular orbital - HRP horseradish peroxidase - LPO lactoperoxidase (EC 1.11.1.7) - LUMO lowest unoccupied molecular orbital     

Molecular responses of plants to an increased incidence of heat shock

Abstract. Climatic change as a result of the greenhouse effect is widely predicted to increase mean temperatures globally and, in turn, increase the frequency with which plants are exposed to heat shock conditions, particularly in the semi-arid tropics. The consequences of extreme high-temperature treatments on plants have been considered, particularly in relation to the synthesis of heat shock proteins (HSPs) and the capacity to acquire thermotolerance. The heat shock response is described using results obtained with seedlings of the tropical cereals, sorghum ( Sorghum bicolor ) and pearl millet ( Pennisetum glaucum ). A gradual temperature increase, as would occur in the field, is sufficient to induce thermotolerance. The synthesis of HSPs is a transient phenomenon and ceases once the stress is released. Despite the persistence of the HSPs themselves, de novo synthesis of HSPs is required for the induction of thermotolerance each time high temperatures are encountered. The effect of a repeated, diurnal heat shock was investigated and genotypic differences found in the ability to induce the heat shock response repeatedly.     

Effects of source-sink relations on photosynthetic acclimation to elevated CO2

Abstract. While photosynthesis of C₃ plants is stimulated by an increase in the atmospheric CO₂ concentration, photosynthetic capacity is often reduced after long-term exposure to elevated CO₂. This reduction appears to be brought about by end product inhibition, resulting from an imbalance in the supply and demand of carbohydrates. A review of the literature revealed that the reduction of photosynthetic capacity in elevated CO₂ was most pronounced when the increased supply of carbohydrates was combined with small sink size. The volume of pots in which plants were grown affected the sink size by restricting root growth. While plants grown in small pots had a reduced photosynthetic capacity, plants grown in the field showed no reduction or an increase in this capacity. Pot volume also determined the effect of elevated CO₂ on the root/shoot ratio: the root/shoot ratio increased when root growth was not restricted and decreased in plants grown in small pots. The data presented in this paper suggest that plants growing in the field will maintain a high photosynthetic capacity as the atmospheric CO₂ level continues to rise.     

An experimental test of the eddy correlation technique over a Mediterranean macchia canopy

Abstract. Flux densities of water vapour and carbon dioxide were measured for a Mediterranean macchia canopy. Results show good agreement between the measured available energy and the sum of latent sensible and heat flux densities determined with the eddy correlation technique. Joint evaluation of the Bowen ratio, aerodynamic resistance, canopy resistance and the 'omega factor' suggests that the macchia canopy is intermediate in aerodynamic roughness between coniferous and deciduous canopies. Maximum daytime carbon flux densities ranged from -14 to -22(μnol m⁻² s⁻¹ on a ground area basis. The ratio of transpiration to assimilation (E/A) was a function of incident photo-synthetic photon flux density below about 400 μmol m⁻²s⁻¹ and above it was fairly constant at 272 mol mol⁻¹ (H₂O/CO₂). The relationship between carbon influx and canopy conductance was linear. Results show promising applications of the eddy correlation technique for evaluating physiological features of canopies, treated as unitary functional systems.