Post-ischaemic silencing of p66Shc reduces ischaemia/reperfusion brain injury and its expression correlates to clinical outcome in stroke.

AIM: Constitutive genetic deletion of the adaptor protein p66(Shc) was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66(Shc) gene regulation in human ischaemic stroke. METHODS AND RESULTS: Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66(Shc) was injected intravenously. We observed that post-ischaemic p66(Shc) knockdown preserved blood-brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66(Shc) preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66(Shc) gene expression is transiently increased and that this increase correlates with short-term neurological outcome. CONCLUSION: Post-ischaemic silencing of p66(Shc) upon reperfusion improves stroke outcome in mice while the expression of p66(Shc) gene correlates with short-term outcome in patients with ischaemic stroke.

Onconeuronal cerebellar degeneration-related antigen, Cdr2, is strongly expressed in papillary renal cell carcinoma and leads to attenuated hypoxic response.

The onconeuronal cerebellar degeneration-related antigen Cdr2 is associated with paraneoplastic syndromes. Neoplastic expression of Cdr2 in ovary and breast tumors triggers an autoimmune response that suppresses tumor growth by developing tumor immunity, but culminates in cerebellar degeneration when Cdr2-specific immune cells recognize neuronal Cdr2. We identified Cdr2 as a novel interactor of the hypoxia-inducible factor (HIF) prolyl-4-hydroxylase PHD1 and provide evidence that Cdr2 might represent a novel important tumor antigen in renal cancer. Strong Cdr2 protein expression was observed in 54.2% of papillary renal cell carcinoma (pRCC) compared with 7.8% of clear-cell RCC and no staining was observed in chromophobe RCC or oncocytoma. High Cdr2 protein levels correlated with attenuated HIF target gene expression in these solid tumors, and Cdr2 overexpression in tumor cell lines reduced HIF-dependent transcriptional regulation. This effect was because of both attenuation of hypoxic protein accumulation and suppression of the transactivation activity of HIF-1alpha. pRCC is known for its tendency to avascularity, usually associated with a lower pathological stage and higher survival rates. We provide evidence that Cdr2 protein strongly accumulates in pRCC, attenuates the HIF response to tumor hypoxia and may become of diagnostic importance as novel renal tumor marker.

Measuring tree-ring increments on tree bole sections with a video-based robotic positioner.

We report on the design and performance of a system that speeds measurement of radial tree-ring increments on tree stem disks; this method replaces the usual binocular microscope with a video image, and automates the measuring and recording processes. The system was used to measure bole sections cut from stems at various heights to determine volume growth of representative trees in an old-growth ponderosa pine stand. The objective of the measurement system was to speed acquisition of annual growth increments from a large number of disks. A personal computer controls the location of a video camera in a 3-axis positioning system. The operator views the sample on a video monitor and positions the camera over each ring by selecting it with a computer-driven mouse. The computer measures and records the distance that the camera moves between each ring. Task selection is facilitated by menu-driven software that also formats, checks and organizes data files. Measurements have a resolution of 0.026 mm; however, finer resolution could be obtained with a different camera lens. Tests of measurement variability (repeated measurements by individual operators on a single radius) indicated standard errors of 0.006 mm or less for the first measurement sets for four operators. Correlation coefficients among four radii per bole section were as low as 0.66 for a whole tree, suggesting that measurements on single radii may provide poor estimates of radial growth for old trees. This system also offers the potential for automatic ring detection and measurement.

A proposed microscopic elastic wave theory for ultrasonic backscatter from myocardial tissue.

The physical structures responsible for ultrasonic scattering from myocardial tissue have not yet been conclusively defined. It is hypothesized in this paper that the backscatter from myocardium is primarily due to inhomogeneities approximately the size of the myocytes. In particular, it is proposed that the acoustic contrast responsible for the scattering is that between the extracellular collagen network that surrounds each myocyte (or myocyte bundle) and the rest of the tissue (the myocytes' intracellular contents). To test this hypothesis, a simple elastic wave scattering model for myocardium was developed. An elementary scatterer is modeled as an ellipsoidal shell, having the material properties of wet collagen, imbedded in a host medium having the average properties of myocardium. The first Born approximation to elastic scattering is used to calculate the frequency-dependent scattering from a single scatterer. To scale up from a single scatterer to a distribution of scatterers, it is assumed that the power received at the transducer is simply the sum of the power scattered in the direction of the transducer by each individual scatterer located in the active volume of the beam (an independent-scatterer approximation). Calculations are restricted to the backscattering direction (pulse-echo), although the theory can accommodate pitch-catch scattering at all angles. With the aid of a computer program, the acoustic backscatter coefficient is calculated using the Born formalism and then measurement effects (frequency-dependent beam width and attenuation correction factors) are incorporated to arrive at calculated integrated (frequency-averaged) backscatter. Both the backscatter coefficient and integrated backscatter are calculated for angles of incidence that range from parallel to the long axis of the scatterer to perpendicular to this fiber direction. For the low MHz frequencies typically used in clinical echocardiography, the calculated absolute magnitude of the acoustic backscatter coefficient lies within a range from 0.0001 to 0.001 cm-1 sr-1. For selected fiber geometries, the anisotropy in integrated backscatter as the angle of incidence is varied with respect to the fiber orientation is about 10 dB. The predicted frequency dependence of the acoustic backscatter coefficient is calculated to be about f3.9 in the low MHz frequency range. These calculated results are reasonably consistent with published experimental measurements and provide a successful preliminary test of the hypothesis.

Evaluating progress toward closed forest models based on fluxes of carbon, water and nutrients.

Closed system models are defined as mathematical models of systems having specified boundaries within which all flows into and out of the system are accounted for. Closure is obtained experimentally when we can measure all the flows and do not depend on residuals. The meeting on which this volume is based discussed a range of models and approaches to modeling, and the possibility of achieving closure. There was general agreement that we can develop closed system models of the water balance, carbon cycle and nutrient fluxes at the stand level. Confidence in our ability to account for all the flows is greatest for water, decreasing progressively for carbon and nutrients. The priority areas for research on the carbon balance are belowground processes, foliage dynamics and respiration. The problems requiring particular attention in relation to the water balance are the measurement of interception losses, lateral flow in the soil and evaporation from snow. Areas warranting particular research attention in relation to nutrient fluxes through forest stands are the rates, and the controls on rates, of nutrient uptake by trees, and rates of mineralization with emphasis on the importance of microbial processes at the ecosystem level. Most models are written for uniform conditions. Forests are not uniform so the problem of heterogeneity, and how to deal with it in models, requires considerable attention, as does the question of how to scale up, to deal with large areas. There are a great many forest models of all types and the continual development of new ones may not be an effective use of research resources. There is a need for some assessment of the range of models currently existing, or under development, and for moves toward a directed strategy of model structure and development.

Report on discussion of paper by J. J. Landsberg.

Unavailable

Report on summary session.

Three participants were asked to contribute to a summary session on the last day of the symposium. They were invited to discuss the three major cycles of external phenomena that affect plant physiological processes; namely, the diurnal cycle, the wetting and drying cycle, and the annual cycle.

An interpretation of some whole plant water transport phenomena.

A treatment of water flow into and through plants to the evaporating surface of the leaves is presented. The model is driven by evaporation from the cell wall matrix of the leaves. The adsorptive and pressure components of the cell wall matric potential are analyzed and the continuity between the pressure component and the liquid tension in the xylem established. Continuity of these potential components allows linking of a root transport function, driven by the tension in the xylem, to the leaf water potential. The root component of the overall model allows for the solvent-solute interactions characteristic of a membrane-bound system and discussion of the interactions of environmental variables such as root temperature and soil water potentials. A partition function is developed from data in the literature which describes how water absorbed by the plant might be divided between transpiration and leaf growth over a range of leaf water potentials.Relationships between the overall system conductance and the conductance coefficients of the various plant parts (roots, xylem, leaf matrix) are established and the influence of each of these discussed.The whole plant flow model coupled to the partition function is used to simulate several possible relationships between leaf water potential and transpiration rate. The effects of changing some of the partition function coefficients, as well as the root medium water potential on these simulations is illustrated.In addition to the general usefulness of the model and its ability to describe a wide range of situations, we conclude that the relationships used, dealing with bulk fluid flow, diffusion, and solute transport, are adequate to describe the system and that analogically based theoretical systems, such as the Ohm's law analogy, probably ought to be abandoned for this purpose.

Leaf Conductance during the Final Season of a Senescing Aspen Branch.

Leaf conductance, transpiration, and environmental conditions were measured on two aspen (Populus tremuloides Michx.) branches in a natural stand, using an automatic cuvette system. Fortuitously, leaves on one branch senesced about 10 days early, allowing comparison between a senescing branch and a normal branch. Terminal bud development was retarded on the senescent branch, and a portion of the branch eventually abscised about 20 centimeters from the end. Roughly 1% to 2% of the other branches on the study tree and adjacent trees of that clone also senesced and were dead the following spring.Although no visual symptoms of senescence were observed until September, stomatal behavior was atypical shortly after leaves were fully expanded. During July and August, leaf conductances under full sunlight were higher on the branch which senesced than on the branch which was normal, reaching values greater than 1.0 centimeters per second, and conductance was highly variable.

Leaf conductance as a function of photosynthetic photon flux density and absolute humidity difference from leaf to air.

FOR AN ENTIRE SEASON OF STOMATAL ACTIVITY, LEAF OR NEEDLE CONDUCTANCE WAS OBSERVED ON FOUR SPECIES, EACH IN A DIFFERENT GENUS: Engelmann spruce (Picea engelmannii Parry ex Engelm.), subalpine fir (Abies lasiocarpa [Hook.] Nutt.), lodgepole pine (Pinus contorta var. latifolia Engelm.), and aspen (Populus tremuloides Michx.). Conductance in the natural environment was described for all species by photosynthetic photon flux density (PPFD) and absolute humidity difference from leaf to air (DAH), as follows: Conductance = b(1) ( radicalPPFD/ radicalDAH) + b(2) ( radicalPPFD/DAH) + b(3) ( radicalPPFD/DAH(2)). The only data not fitting this relationship were conifer data collected after freezing nights or aspen data collected during a short period in August when water stress occurred. In both cases, leaf conductance was reduced. It is proposed that PPFD and DAH are primary factors controlling stomatal function for plants growing in their native range; secondary factors, such as temperature and water stress, affect conductance intermittently, except when plants are growing outside their normal environmental conditions.

Evaluation of season, temperature, and water stress effects on stomata using a leaf conductance model.

A model was developed earlier describing conductance for three conifers (Picea engelmannii Parry ex Engelm., Abies lasiocarpa [Hook.] Nutt., and Pinus contorta var. latifolia Engelm.) and one hardwood (Populus tremuloides Michx.) using only two terms, photosynthetic photon flux density (PPFD) and absolute humidity difference from leaf to air (DAH). Using residual analysis techniques (actual minus estimated conductance), it was determined that no seasonal or temperature effects existed that were not taken into account with PPFD and DAH. However, conductance was reduced on days following cold nights (below 4 degrees C) or, in aspen, when xylem pressure potential was below -20 bars (1 bar = 10(5) Pa). The following model takes these terms into account: Conductance = b(1) ( radicalPPFD/ radicalDAH) + b(2) ( radicalPPFD/DAH) + b(3) ( radicalPPFD/DAH(2)) + b(4)f(T(min)) + b(5)f(psi(threshold)), where the first three terms describe normal conductance, and the last two terms account for reductions in conductance caused by cold night temperatures or water stress.

Stomatal response of engelmann spruce to humidity, light, and water stress.

Stomatal response of Engelmann spruce (Picea engelmannii Engelm.) to environmental conditions was studied in the natural subalpine environment and under controlled laboratory conditions. Stomata of naturally occurring trees responded to the difference in absolute humidity from leaf to air. When foliage was exposed to full sunlight, stomatal conductance decreased as the absolute humidity difference increased. In the shade, where photosynthetically active radiation was 10% of that in full sunlight, stomatal closure at large absolute humidity differences was much more complete. No effect of soil or air temperatures on stomatal aperture was observed in the field, nor were differences among three contrasting sites detected. Under growth chamber conditions, stomata responded to photosynthetically active radiation, but conductances were influenced by leaf-to-air differences in absolute humidity. Leaf water potentials below - 15 bars resulted in lower conductances over a range of humidity and light conditions. Because net photosynthesis under shaded conditions in the natural environment must be very low, stomatal closure could result in considerable savings in water while having a minimum effect on net photosynthesis.

Leaf water stress in engelmann spruce: influence of the root and shoot environments.

The response of xylem pressure potential of Engelmann spruce (Picea engelmannii Engelm.) to environmental factors was studied in the natural subalpine environment. Data were analyzed in the context of a leaf water potential model based upon the van den Honert model for water transport through the soil-plant-atmosphere continuum. At soil temperatures of 10 to 15 C, xylem pressure potential decreased to about -10 bars as the ratio of leaf to air absolute humidity difference to leaf diffusion resistance (an estimate of transpiration) increased. The potentials were slightly lower at all flux rates above zero when the soil temperature was 5 to 10 C, and at temperatures of 0 to 5 C the potentials decreased sharply to as low as -20.4 bars, even though the soil water supply was adequate. The relative viscosity of water and soil to leaf resistances for flow were compared for Engelmann spruce and citrus at low soil temperatures. These comparisons indicated that decreased root permeability was probably not an important factor causing higher stresses in spruce at 5 to 10 C, but for citrus, root permeability became limiting at soil temperatures as high as 13.5 C. Xylem pressure potential was correlated with net radiation during the daytime when soil temperature was above 7 C. Under other conditions, however, xylem potential and net radiation apparently had a different relationship. The relationship between flux density and potential was the same on unshaded and shaded portions of the crown, with differences in potential related to differences in flux density.

Stomatal Response to Environment with Sesamum indicum. L.

Leaf resistance of Sesamum indicum L. increased when the humidity gradient between leaf and air was increased, at moderate temperatures, even though calculated carbon dioxide concentrations within the leaf decreased slightly. Mesophyll resistance remained relatively constant when humidity gradients were changed, indicating that the increases in leaf resistance were mainly caused by reductions in stomatal aperture and that nonstomatal aspects of photosynthesis and respiration were not affected. Low carbon dioxide concentrations inside the leaf decreased but did not eliminate resistance response to the humidity gradient. Internal carbon dioxide concentrations had little effect on resistance in humid air but had moderate effects on resistance with large humidity gradients between leaf and air. Stomatal response to humidity was not present at high leaf temperatures. Effects of humidity gradients on photosynthetic and stomatal responses to temperature suggested that large humidity gradients may contribute to mid-day closure of stomata and depressions in photosynthesis.

Efficiency and regulation of water transport in some woody and herbaceous species.

The efficiency with which plants transport water is related to the water potential differences required to drive water fluxes from the soil to the leaf. A comparative study of two woody and three herbaceous species (Citrus sinensis L. cv. Koethen, Pyrus kawakami L., Helianthus annuus L. cv. Mammoth Russian, Capsicum frutescens L. cv. Yolo Wonder, and Sesamum indicum L. cv. Glauca) indicated contrasts in water transport efficiency. Depression of leaf water potential in response to transpiration increases was found in the woody species; the herbaceous species, however, had more efficient water transport systems and presented no measurable response of leaf water potential to transpiration changes. Different maximum transpiration rates under the same climatic conditions were observed with different species and may be accounted for by stomatal response to humidity gradients between leaf and air. Leaf diffusion resistance in sesame increased markedly as the humidity gradient was increased, while leaf resistance of sunflower responded less to humidity. Stomata appeared to respond directly to the humidity gradient because changes in leaf water potential were not detected when leaf resistance increased or decreased.

The osmotic potential of polyethylene glycol 6000.

Osmotic potential (psi(s)) of aqueous solutions of polyethylene glycol 6000 (PEG-6000) was curvilinearly related to concentration. At given concentrations, psi(s) increased linearly with temperature. The effects of concentration and temperature on psi(s) of PEG-6000 solutions differ from those for most salts and sugars and apparently are related to structural changes in the PEG polymer. Measurements of psi(s) with thermocouple psychrometers are more negative than those with a vapor pressure osmometer, with the psychrometer probably giving the more nearly correct psi(s) for bulk solutions. An empirical equation permits calculation of psi(s) from known concentrations of PEG-6000 over a temperature range of 15 to 35 C. Viscometery and gravimetric analysis are convenient methods by which the concentrations of PEG-6000 solutions may be measured.

Evaluation of water stress control with polyethylene glycols by analysis of guttation.

The water relations of pepper plants (Capsicum frutescens L.) under conditions conducive to guttation were studied to evaluate the control of plant water stress with polyethylene glycols. The addition of polyethylene glycol 6000 to the nutrient solution resulted in water relations similar to those expected in soil at the same water potentials. Specifically, xylem pressure potential in the root and leaf became more negative during a 24-hour treatment period, while osmotic potential of the root xylem sap remained constant. The decrease in pressure potential was closely correlated with the decrease in osmotic potential of the nutrient solution. In contrast, the addition of polyethylene glycol 400 to the nutrient medium resulted in a reduction of osmotic potential in the root xylem sap; this osmotic adjustment in the xylem was large enough to establish an osmotic gradient for entry of water and cause guttation at a nutrient solution osmotic potential of -4.8 bars. Pressure potential in the root and leaf xylem became negative only at nutrient solution osmotic potentials lower than -4.8 bars. About half of the xylem osmotic adjustment in the presence of polyethylene glycol 400 was caused by increased accumulation of K(+), Na(+), Ca(2+), and Mg(2+) in the root xylem. These studies indicate that larger polyethylene glycol molecules such as polyethylene glycol 6000 are more useful for simulating soil water stress than smaller molecules such as polyethylene glycol 400.

Extensibility of pericarp tissue in growing citrus fruits.

The tensile force existing in the pericarp of a growing citrus (Citrus sinensis) fruit 17 to 19 centimeters in circumference was sufficiently high to cause a 3% shrinkage of the pericarp when it was excised. When a fruit was cut along the equator to the central axis, shrinkage of the pericarp resulted in the formation of a wedge-shaped gap at the cut. Stretch modulus of the pericarp was determined by measuring the force required to stretch excised strips of tissue to 1% longer than their excised length. Measurements were made on successive layers of pericarp tissue 5 millimeters wide and 1 millimeter thick taken from the fruit equator. All layers required more force for extension at lower temperatures and high water potentials than at high temperatures and low water potentials. The stretch modulus ranged from 0.88 to 2.16 kilograms per square millimeter depending upon the layer, temperature, and water potential. The inner layers, consisting primarily of mesocarp, had stretch moduli only 60 to 70% as great as the outer layer which consisted of exocarp tissue. Measurements of the stretch modulus of tissues from the pericarp support the hypothesis that changes in the tension existing in the pericarp depend upon conditions in the pericarp and are not related to changes in volume or pressure in the juice vesicles.

Water potential components in growing citrus fruits.

Growing navel orange fruits (Citrus sinensis) 5.4 to 5.7 centimeters in diameter were used as a model system to determine the effects of transpiration and carbohydrate translocation on water and osmotic potentials in fruit tissues. Evidence supported the hypothesis that osmotic potential in the vesicles would be affected little by changes in transpiration or carbohydrate translocation because the vesicles are anatomically isolated from the transpiration stream and are at the end of the carbohydrate translocation pathway. In the mesocarp tissue, which contains a vascular network, osmotic potential decreased during the daytime when environmental conditions favored transpiration and increased at night. Exocarp water potential followed a similar pattern. Girdling of the stem above the fruits 5 days before sampling caused an increase of osmotic potential in the mesocarp but had no effect on exocarp water potential. Neither diurnal changes in transpiration nor girdling of the stem affected the osmotic potential of the vesicles.Osmotic potentials in all tissues of the fruit were in the range of -10 to -15 bars. Measurements of osmotic potential at 16 locations along a longitudinal plant through the fruit axis showed that osmotic potential increased from the stem to the stylar end, but it decreased from the pericarp tissues to the vesicles. As exocarp water potential decreased during a 20-day period after watering, osmotic potential decreased in the vesicles and exocarp. Turgor pressure, calculated as the difference between water and osmotic potentials, decreased with water potential in the vesicles but not in the exocarp. The lack of decrease of turgor pressure in the exocarp may result from a measurement error caused by pectins or from osmotic adjustment related to carbohydrate accumulation at low water potentials.

Water relations of pine seedlings in relation to root and shoot growth.

The effects of water stress on growth and water relations of loblolly and white pine seedlings were studied during series of drying cycles. As mean soil water potential decreased, growth of roots, needles, and buds decreased. Growth of roots during successive severe drying cycles was not uniform, however. A study of needle and root extension showed that of the total growth of roots for 3 7-day drying cycles, only 6% occurred during the third cycle, while needle extension was uniform for the 3 cycles. The difference in response of needles and roots to drying cycles may be attributed primarily to the effect of water stress on the growing region. When subjected to a severe stress, roots matured toward the tip and became dormant, resulting in less growth during subsequent drying cycles. The intercalary growing region of needles, however, was not altered seriously enough by the stress to cause a difference in amount of growth during each drying cycle.Transpiration of loblolly pine was lower in the second drying cycle than in the first. Needle water potential after rewatering was as high as that of control plants watered daily; root resistance was apparently not important in restricting transpiration during a second drying cycle. Needle diffusion resistance of loblolly pine, measured with a low-resistance diffusion porometer, was slightly higher during the second drying cycle than during the first. In addition, many primary needles were killed during the first period of stress. These factors contributed to the reduction of transpiration during the second drying cycle. Diffusion resistance of Coleus increased and transpiration ceased during the first drying cycle while water potential remained relatively high. After rewatering, both leaf resistance and transpiration returned to the control level, presumably because the stress during the first period of drying was not severe. The diffusion resistances observed for well-watered plants were 30 to 50 sec.cm(-1) for loblolly pine, 3 to 5 sec.cm(-1) for Coleus, and 4 to 6 sec.cm(-1) for tomato. These values agree closely with those reported by other workers.