Comprehensive messenger ribonucleic acid profiling reveals that peroxisome proliferator-activated receptor gamma activation has coordinate effects on gene expression in multiple insulin-sensitive tissues.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists, including the glitazone class of drugs, are insulin sensitizers that reduce glucose and lipid levels in patients with type 2 diabetes mellitus. To more fully understand the molecular mechanisms underlying their therapeutic actions, we have characterized the effects of the potent, tyrosine-based PPAR gamma ligand GW1929 on serum glucose and lipid parameters and gene expression in Zucker diabetic fatty rats. In time-course studies, GW1929 treatment decreased circulating FFA levels before reducing glucose and triglyceride levels. We used a comprehensive and unbiased messenger RNA profiling technique to identify genes regulated either directly or indirectly by PPAR gamma in epididymal white adipose tissue, interscapular brown adipose tissue, liver, and soleus skeletal muscle. PPAR gamma activation stimulated the expression of a large number of genes involved in lipogenesis and fatty acid metabolism in both white adipose tissue and brown adipose tissue. In muscle, PPAR gamma agonist treatment decreased the expression of pyruvate dehydrogenase kinase 4, which represses oxidative glucose metabolism, and also decreased the expression of genes involved in fatty acid transport and oxidation. These changes suggest a molecular basis for PPAR gamma-mediated increases in glucose utilization in muscle. In liver, PPAR gamma activation coordinately decreased the expression of genes involved in gluconeogenesis. We conclude from these studies that the antidiabetic actions of PPAR gamma agonists are probably the consequence of 1) their effects on FFA levels, and 2), their coordinate effects on gene expression in multiple insulin-sensitive tissues.

Changes in stomatal behaviour in the calcicole Leontodon hispidus due to the disruption by ozone of the regulation of apoplastic Ca2+ by trichomes.

Regulation of the concentration of Ca2+ in the apoplast of plants is essential in order to allow Ca(2+)-dependent processes, such as Ca(2+)-mediated signal transduction in stomatal guard cells, to function correctly. This is particularly important for plants growing with high levels of Ca2+ in the rhizosphere. Recently, we have shown that in two calcicoles, Leontodon hispidus L. and Centaurea scabiosa L., trichomes play a key role in this regulatory process. Ozone is known to have a marked effect on plant Ca2+ homeostasis. Therefore, we have examined the effect of this pollutant on the regulation by trichomes of apoplastic Ca2+ in the calcicole L. hispidus. Treatment with 100 nl l-1 ozone resulted in a reduction in stomatal conductance of approximately 25% in plants grown with 15 mM Ca2+ in the rhizosphere. Analysis of total Ca2+ levels revealed that these changes in stomatal behaviour reflect a decrease in the ability of trichomes to sequester Ca2+. The amount of Ca2+ present in the trichome tip cell was reduced by approximately 38%. This was accompanied by an increase in the levels of Ca2+ in the guard cells and other tissues of the leaf. These data suggest that ozone has a detrimental effect on the ability of trichomes to regulate the concentration of apoplastic Ca2+ in L. hispidus, resulting in altered stomatal behaviour, and hence gaseous exchange, possibly due to the disruption of guard-cell Ca(2+)-mediated signal transduction. This has important implications for the growth and survival of plants growing in Ca(2+)-rich soils.

The role of apolipoprotein E in Alzheimer's disease: pharmacogenomic target selection.

The association of inheritance of different apolipoprotein E (APOE, gene; apoE, protein) alleles with the risk and rate of onset of Alzheimer's disease (AD) is now well established and widely confirmed. While there are now a collection of hypotheses concerning the specific relationship of APOE polymorphisms to various phenotypic manifestations of AD, no single compelling theory has been tested and universally accepted. The only clear fact emerging during the past 6 years is that differences in APOE genotype affect the average rate of disease onset as a predictable function of the inheritance of this polymorphic gene. Methods now exist to enable experimental designs to study the metabolic effects of inheriting different APOE alleles, addressing what differences that may be present for many years, perhaps over the entire lifetime, can lead to earlier or later manifestations of the disease and are therapeutically tractable. This review summarizes part of an experimental approach to identify biological pathways influenced by the different APOE polymorphisms that are relevant to the pathogenesis of AD.

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.

Stomata and plant water relations: does air pollution create problems?

Small changes in the gaseous composition of the atmosphere have many different impacts on terrestrial plants. Some of the most important involve changes in stomatal control of leaf conductance. Evolution has provided highly complex mechanisms by which stomata respond to a wide range of environmental factors to balance the conflicting priorities of carbon gain for photosynthesis and water conservation. These mechanisms involve direct responses of the guard cells to aspects of the aerial environment, and hormonal communication within the plant enabling conductance to be adjusted according to soil moisture status. Various aspects of these delicately balanced mechanisms can be disturbed by air pollutants. Impairment of the regulation of plant water use by SO2 and O3 has been known for some years, but there are still many obstacles to our understanding of the variations in response between species, or even between genotypes of the same species. A surprising outcome of some recent studies is the suggestion that CO2 pollution may disrupt the control of water relations in some species because their stomata do not close sufficiently in CO2-enriched air. It has often been taken for granted that the elevation of atmospheric CO2 would lead to economies in water use by plant canopies, but the underlying assumptions are now being seriously questioned.

Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III.

Analysis of patients with inherited hypokalaemic alkalosis resulting from salt-wasting has proved fertile ground for identification of essential elements of renal salt homeostasis and blood-pressure regulation. We now demonstrate linkage of this phenotype to a segment of chromosome 1 containing the gene encoding a renal chloride channel, CLCNKB. Examination of this gene reveals loss-of-function mutations that impair renal chloride reabsorption in the thick ascending limb of Henle's loop. Mutations in seventeen kindreds have been identified, and they include large deletions and nonsense and missense mutations. Some of the deletions are shown to have arisen by unequal crossing over between CLCNKB and the nearby related gene, CLCNKA. Patients who harbour CLCNKB mutations are characterized by hypokalaemic alkalosis with salt-wasting, low blood pressure, normal magnesium and hyper- or normocalciuria; they define a distinct subset of patients with Bartter's syndrome in whom nephrocalcinosis is absent. These findings demonstrate the critical role of CLCNKB in renal salt reabsorption and blood-pressure homeostasis, and demonstrate the potential role of specific CLCNKB antagonists as diuretic antihypertensive agents.

Multilocus linkage of familial hyperkalaemia and hypertension, pseudohypoaldosteronism type II, to chromosomes 1q31-42 and 17p11-q21.

Essential hypertension is a common multifactorial trait. The molecular basis of a number of rare diseases that after blood pressure in humans has been established, identifying pathways that may be involved in more common forms of hypertension. Pseudohypoaldosteronism type II (PHAII, also known as familial hyperkalaemia and hypertension or Gordon's syndrome; OMIM #145260), is characterized by hyperkalaemia despite normal renal glomerular filtration, hypertension and correction of physiologic abnormalities by thiazide diuretics. Mild hyperchloremia, metabolic acidosis and suppressed plasma renin activity are variable associated findings. The pathogenesis of PHAII is unknown, although clinical studies indicate an abnormality in renal ion transport. As thiazide diuretics are among the most efficacious agents in the treatment of essential hypertension, understanding the pathogenesis of PHAII may be of relevance to more common forms of hypertension. Analysis of linkage in eight PHAII families showing autosomal dominant transmission demonstrates locus heterogeneity of this trait, with a multilocus lod score of 8.1 for linkage of PHAII to chromosomes 1q31-q42 and 17p11-q21. Interestingly, the chromosome-17 locus overlaps a syntenic interval in rat that contains a blood pressure quantitative trait locus (QTL). Our findings provide a first step toward identification of the molecular basis of PHAII.

Root production and turnover and carbon budgets of two contrasting grasslands under ambient and elevated atmospheric carbon dioxide concentrations.

Monoliths of two contrasting vegetation types, a species-rich grassland on a brown earth soil over limestone and species-poor community on a peaty gley, were transferred to solardomes and grown under ambient (350 µ 1-1 ) and elevated (600 µ11-1 ) CO2 for 2 yr. Shoot biomass was unaltered but root biomass increased by 40-50% under elevated CO2 . Root production was increased by elevated CO2 in the peat soil, measured both as instantaneous and cumulative rates, but only the latter measure was increased in the limestone soil. Root growth was stimulated more at 6 cm depth than at 10 cm in the limestone soil. Turnover was faster under elevated CO2 in the peat soil, but there was only a small effect on turnover in the limestone soil. Elevated CO2 reduced nitrogen concentration in roots and might have increased mycorrhizal colonization. Respiration rate was correlated with N concentration, and was therefore lower in roots grown at elevated CO2 . Estimates of the C budget of the two communities, based upon root production and on net C uptake, suggest that C sequestration in the peat soil increases by c. 0.2 kg C m -2 yr-1 (= 2 t ha yr-1 ) under elevated CO2 .

Changes in Stomatal Behavior and Guard Cell Cytosolic Free Calcium in Response to Oxidative Stress.

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10-5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10-5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.

DCC expression is altered by multiple mechanisms in brain tumours.

The DCC (deleted in colorectal cancer) candidate tumor suppressor gene spans greater than 1350 kilobases at chromosomes 18q21.1 and encodes a transmembrane protein of unknown function. Although DCC is expressed in a number of adult tissues, its expression is highest in the brain and we have, therefore, undertaken studies to determine if DCC inactivation might contribute to tumors arising there. Decreased or absent DCC protein expression was noted in more than 50% of the thirty brain tumors studied. Although specific mutations in the DCC gene were not identified, a variety of mechanisms appeared to contribute to the altered DCC expression, including allelic loss, aberrant splicing of transcripts and allele-specific loss of transcripts. In total, the data suggest that DCC inactivation may be important in brain tumor pathogenesis.

Nitrogen, phosphorus and potassium uptake and demand in Agrostis capillaris: the influence of elevated CO2 and nutrient supply.

Responses to elevated CO2 have been studied using Agrostis capillaris L., an upland grass which is abundant on nutrient-poor soils. Plants were grown in sand culture with a wide range of nitrogen, phosphorus and potassium concentrations, and the impact of CO2 on the demand for nutrients was determined using isotopic root bioassays. Plants grown with the smallest concentrations of N and P showed typical foliar symptoms associated with deficiency of these elements. However, even when supplies of N and P were limiting to growth, additional CO2 (250 ppm above ambient) influenced neither total N nor total P in above-ground tissues, nor nutrient demands as indicated by the bioassay. The estimates of the demand of the plants for K from the 86 Rb bioassay indicated an appreciable increase when plants were raised in elevated CO2 . For plants of the same size with the same nutrient supply, those grown in elevated CO2 consistently displayed an increased internal demand for K. Uptake of K was not however, enhanced by elevated CO2 even in non-limiting conditions and it might therefore be limited by a factor other than K supply. The overall conclusion from the experiments is that when A. capillaris is grown in elevated CO2 , uptake of N, P and K fails to increase proportionally with dry mass. This was true even when nutrient supplies were adequate, and it appears that nutrient-use-efficiency might increase to enable the plants to maintain growth in elevated CO2 .

Effects of exposure to ozone and water stress on the following season's growth of beech (Fagus sylvatica L.).

Bud break and growth of beech trees were measured during the growing season of 1992. The trees had previously been exposed, through the growing season of 1991, to one of four treatments: (1) episodic ozone for 128 d, total dose = 46-3 ppm h; (2) air purified by filtration through Purified and charcoal; (3) ozone together with a period of water shortage: (4) filtered air with a period of water shortage. Despite uniform growth conditions during 1992. the previous water stress treatment caused bud break to begin slightly earlier, but the rates of shoot growth and the length of the first flush branches were reduced (40%). In well-watered plants ozone caused a reduced Tate of shoot growth during the first week after bud break. The total amount of growth during 1992 was shown to be reduced (36%) by the previous water stress treatment, In the case of the well-watered trees, exposure to ozone in the previous year reduced the amount of new growth by 17%, which was the result of reduced internal expansion. When ozone was combined with water stress, there was no further reduction in the amount of new growth, but there was a fall in the number of internodes relative to water-stressed plants grown in filtered air. Ozone and water stress applied singly reduced growth. A combination of the two stresses, however, caused no additional reduction in growth, but reduced the number of internodes. The possible implications of the aftereffects of these stresses are discussed in relation to reduced productivity and canopy structure.

Low temperature-enhanced inhibition of photosynthesis by oxides of nitrogen in lettuce (Lactuca sativa L.).

The response of photosynthetic gas exchange to oxides of nitrogen (NOx) was studied in leaves of lettuce (Lactuca sativa L.) at different temperatures. Exposure to > high concentrations (e.g. 13 µmol NOx mol-1 ), similar to those often found in commercial glasshouses, caused a rapid inhibition of the net assimilation of CO2 . This appeared to be by a direct effect on photosynthesis rather than by a change in the stomatal conductance. In ambient CO2 , (345 µmol mol-1 ), the percentage inhibition at 10 and 5°C was approximately 3× and 5×, respectively, that measured at 20°C. This effect of temperature also occurred when measured in CO2 , enriched air (1050 µmol mol-1 ), which would normally accompany NOx in a glasshouse. The extent of photosynthetic inhibition caused by NOx was, however, always less in high than in low CO2 . The results suggest that when burning fuel to raise the CO2 , concentration and heat the glasshouse air, growers should avoid generating high concentrations of NOx , in conditions of low temperature.

The role of arginine decarboxylase in modulating the sensitivity of barley to ozone.

Polyamines (PA) are known to be involved in the areas of plant physiology and biochemistry which are related to the response of a plant to air pollution. This study examines the role of arginine decarboxylase (ADC), an important rate-limiting enzyme in polyamine synthesis, in barley plants exposed to ozone (O(3)). The activity of ADC increased significantly in O(3)-treated leaves when visible injury was hardly apparent. The increase in ADC activity may be a mechanism to increase the PA levels in O(3)-treated leaves and so minimize the damaging effects of O(3). Supporting this, foliar applications of DL-alpha-difluoromethylarginine (DFMA), a specific inhibitor of ADC, prevented the rise in ADC activity and visible injury was considerable on exposure to O(3). This damage was not due to the foliar sprays, as little visible injury was seen in leaves in the O(3)-free controls. The results are discussed in terms of the roles of PA in conferring O(3) resistance in plants.

The sensitivity of early 20th century cultivars of wheat to air pollution.

The effects of a mixture of SO(2) and NO(2) at a concentration of 0.08 to 0.10 ppm (in microg m(-3): 164-205 for NO(2); 229-286 for SO(2)) were tested for four weeks on two old varieties of winter wheat, Little Joss and Holdfast, introduced in 1908 and 1938, respectively, grown in simulated autumn conditions. After two weeks, root dry weights of both varieties were significantly decreased but, although there was some leaf necrosis, shoot dry weights were unaltered. After a further two weeks, the dry weights of the shoots as well as the roots were significantly decreased. These effects, amounting to a combined 40% decrease in total dry weight, were greater than those in identical experiments carried out with the present day variety Avalon, in which the decrease was 20%. The results suggest that the modern cultivar Avalon is more tolerant of SO(2) and NO(2) than Little Joss and Holdfast, which were grown many years ago.

Effects of sulphur dioxide and nitrogen dioxide on the control of water loss by birch (Betula spp.).

The effects of SO2 and NO2 on the control of water loss by birch trees were investigated using clonal populations of both Betula pendula Roth, (silver birch) and Betula pubescens Khr. (downy birch). Plants were grown in controlled environments and were fumigated in a 2 × 2 factorial experiment with 65 nl l-1 SO2 and/or NO2 , or with equal concentrations of both SO2 and NO2 in four treatments, namely zero, 20, 40 and 60 nl l-1 . Excised leaves showed in increased rate of water loss approximately corresponding to the concentration of pollutant to which they had been previously exposed. When petroleum jelly was applied to the abaxial epidermal surface of polluted leaves the increased rate of water loss was not found, strongly implying that the damage had occurred only at this site, and not on the adaxial epidermis, which is virtually devoid of stomata. Measurements of gas exchange were made on B. pubescens exposed to the four doses of the mixture of the two gases. The rate of transpiration was approximately doubled as a result of the two higher pollution treatments, judging from measurements made later in clean air. When transpiration was measured in high and low CO2 concentrations, polluted plants had a slightly reduced and more variable response to the change in CO2 concentration. Both clones were also exposed first to the four doses of the gas mixture, and then to drought. There was a more rapid onset of water stress, accompanied by an earlier decrease in effective leaf area in the polluted plants, when compared with the unpolluted plants. The abaxial surface of frozen hydrated leaves of the Betula pubescens clone were examined at low temperature with a scanning electron microscope. Damaged epidermal cells were clearly visible on the polluted leaves and thought to be responsible for areas of wide open stomata. It is concluded that leaves of these species are less efficient in the utilization of water after a period of exposure to pollution and are less able to restrict water loss in a time of shortage.

Effect of SO2 on the reproduction of pea aphids, Acyrthosiphon pisum, and the impact of SO2 and aphids on the growth and yield of peas.

Pea aphids feeding from birth to maturity on pea plants (Pisum sativum) exposed to SO(2) concentrations of 50 nl litre(-1) or 80 nl litre(-1) showed a significant 19% increase in the rate of nymph production during the reproductive period, compared to control aphids feeding on plants in charcoal-filtered air. The higher nymph production resulted in a mean 4.6% increase in the intrinsic rate of population increase (rm). In longer term glasshouse fumigation experiments pea aphid populations were, on average, 1.8 times greater on pea plants in ambient air plus 45 nl litre(-1) SO(2) than in ambient air alone. Aphid infestation in ambient air caused a 42% reduction in pea yield and affected most plant parameters adversely. Ambient air plus SO(2) had no direct effect on yield, but, in combination with aphid infestation, a further 10% reduction in yield was recorded.

A large-scale fumigation system for investigating interactions between air pollution and cold stress on plants.

A new large-scale closed chamber fumigation system with cooling facilities is described for studying effects of low concentrations of SO(2), NO(2) and O(3) and low temperatures on woody species and herbaceous plants. The system is based on modified hemispherical greenhouses with a forced air ventilation system. This provides a chamber environment with low spatial variability of pollutant gas concentrations and rapid air circulation which allows exposure of plants at near ambient temperatures and relative humidity. Large capacity cooling units come into operation when ambient temperatures fall below 0 degrees C, and these allow chamber temperatures to be lowered by an additional 4 to 8 degrees C in experiments designed to test whether exposure to pollutants enhances the frost sensitivity of plants.

THE ROLE OF ABSCISIC ACID AND CALCIUM IN DETERMINING THE BEHAVIOUR OF ADAXIAL AND ABAXIAL STOMATA.

Experiments are described which attempted to clarify the reasons for differences in stomatal opening on adaxial and abaxial epidemics. Previous studies had suggested that endogenous auxin levels might be responsible. It was found that adaxial stomata were more sensitive than abaxial stomata to externally supplied abscisic acid (ABA). It is suggested that differing endogenous concentrations of calcium ions in the upper and lower epidermes may contribute to the observed differences in sensitivity to ABA. It is further suggested that studies of stomata on isolated epidermis may represent a useful model system for examining the role of cell or tissue sensitivity in determining the responses to ABA and other hormones. Data are presented which lend support to the suggestion that calcium ions play an important part. Treatment with lanthanum ions, which block calcium channels in cell membranes, had little effect on abaxial stomata, but caused the apertures of adaxial stomata to increase considerably. The chelating agent EGTA eliminated the differential behaviour of adaxial and abaxial stomata, which clearly suggests that calcium ions have a dominant role. Experiments in which EGTA and fusicoccin were presented separately and in combination indicated that their effects were independent and additive. It is suggested that they operate at different locations, or on different processes in the guard cells.