Showing papers by "University of Dundee published in 1999"

Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress

Abstract: Increases in the intracellular levels of reactive oxygen species (ROS), frequently referred to as oxidative stress, represents a potentially toxic insult which if not counteracted will lead to membrane dysfunction, DNA damage and inactivation of proteins. Chronic oxidative stress has numerous pathological consequences including cancer, arthritis and neurodegenerative disease. Glutathione-associated metabolism is a major mechanism for cellular protection against agents which generate oxidative stress. It is becoming increasingly apparent that the glutathione tripeptide is central to a complex multifaceted detoxification system, where there is substantial inter-dependence between separate component members. Glutathione participates in detoxification at several different levels, and may scavenge free radicals, reduce peroxides or be conjugated with electrophilic compounds. Thus, glutathione provides the cell with multiple defences not only against ROS but also against their toxic products. This article discusses how glutathione biosynthesis, glutathione peroxidases, glutathione S-transferases and glutathione S-conjugate efflux pumps function in an integrated fashion to allow cellular adaption to oxidative stress. Co-ordination of this response is achieved, at least in part, through the antioxidant responsive element (ARE) which is found in the promoters of many of the genes that are inducible by oxidative and chemical stress. Transcriptional activation through this enhancer appears to be mediated by basic leucine zipper transcription factors such as Nrf and small Maf proteins. The nature of the intracellular sensor(s) for ROS and thiol-active chemicals which induce genes through the ARE is described. Gene activation through the ARE appears to account for the enhanced antioxidant and detoxification capacity of normal cells effected by many cancer chemopreventive agents. In certain instances it may also account for acquired resistance of tumours to cancer chemotherapeutic drugs. It is therefore clear that determining the mechanisms involved in regulation of ARE-driven gene expression has enormous medical implications.

The p53 pathway

Abstract: Abnormalities of the p53 tumour suppressor gene are among the most frequent molecular events in human and animal neoplasia. Moreover, p53 is one of the most studied proteins in the whole of contemporary biology, with more than 12,500 papers so far written! In this review the choice has been deliberately made not to be fully comprehensive in the coverage of the huge p53 literature. Rather attention is focused on a small number of recent developments which are reviewed in the context of modern models of p53 function. Progress in the analysis of signalling to p53 including phosphorylation cascades, and interactions with proteins such as mdm2 and ARF are highlighted. The plethora of protein-protein interactions is discussed, as are the strategies for defining downstream targets of p53. Finally, the emerging biology of p53 homologues is considered. The need for bridging the gap between reductionist, biochemical and biophysical studies and biological and genetic analysis is emphasized. Only this will provide the needed framework for utilizing the information in clinical care.

Regulation of JNK signaling by GSTp

Abstract: Studies of low basal Jun N-terminal kinase (JNK) activity in non-stressed cells led us to identify a JNK inhibitor that was purified and identified as glutathione S-transferase Pi (GSTp) and was characterized as a JNK-associated protein. UV irradiation or H2O2 treatment caused GSTp oligomerization and dissociation of the GSTp-JNK complex, indicating that it is the monomeric form of GSTp that elicits JNK inhibition. Addition of purified GSTp to the Jun-JNK complex caused a dose-dependent inhibition of JNK activity. Conversely, immunodepleting GSTp from protein extracts attenuated JNK inhibition. Furthermore, JNK activity was increased in the presence of specific GSTp inhibitors and a GSTp-derived peptide. Forced expression of GSTp decreased MKK4 and JNK phosphorylation which coincided with decreased JNK activity, increased c-Jun ubiquitination and decreased c-Jun-mediated transcription. Co-transfection of MEKK1 and GSTp restored MKK4 phosphorylation but did not affect GSTp inhibition of JNK activity, suggesting that the effect of GSTp on JNK is independent of the MEKK1-MKK4 module. Mouse embryo fibroblasts from GSTp-null mice exhibited a high basal level of JNK activity that could be reduced by forced expression of GSTp cDNA. In demonstrating the relationships between GSTp expression and its association with JNK, our findings provide new insight into the regulation of stress kinases.

AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes.

Abstract: Adenosine 5′-monophosphate-activated protein kinase (AMPK) now appears to be a metabolic master switch, phosphorylating key target proteins that control flux through metabolic pathways of hepatic k...

Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation.

Abstract: Growth factor induced activation of phosphoinositide 3-kinase and protein kinase B (PKB) leads to increased activity of the mammalian target of rapamycin (mTOR). This subsequently leads to increased phosphorylation of eIF4E binding protein-1 (4EBP1) and activation of p70 ribosomal S6 protein kinase (p70(S6K)), both of which are important steps in the stimulation of protein translation. The stimulation of translation is attenuated in cells deprived of amino acids and this is associated with the attenuation of 4EBP1 phosphorylation and p70(S6K) activation. It has been suggested that PKB regulates mTOR function by phosphorylation although direct phosphorylation of mTOR by PKB has not been demonstrated previously. In the present work, we have found that PKB directly phosphorylates mTOR and, using phosphospecific antibodies, we have shown this phosphorylation occurs at Ser(2448). Insulin also induces phosphorylation on Ser(2448) and this effect is blocked by wortmannin but not rapamycin, consistent with the effect being mediated by PKB. Amino-acid starvation rapidly attenuated the reactivity of the Ser(2448) phosphospecific antibody with mTOR and this could not be restored by either insulin stimulation of cells or incubation with PKB in vitro. Our findings demonstrate that mTOR is a direct target for PKB and support the conclusion that regulation of phosphorylation of Ser(2448) is a point of convergence for the counteracting regulatory effects of growth factors and amino acid levels.

Systemic Adverse Effects of Inhaled Corticosteroid Therapy: A Systematic Review and Meta-analysis

Abstract: Objective To appraise the data on systemic adverse effects of inhaled corticosteroids. Methods A computerized database search from January 1, 1966, through July 31, 1998, using MEDLINE, EMBASE, and BIDS and using appropriate indexed terms. Reports dealing with the systemic effects of inhaled corticosteroids on adrenal gland, growth, bone, skin, and eye, and reports on pharmacology and pharmacokinetics were reviewed where appropriate. Studies were included that contained evaluable data on systemic effects in healthy volunteers as well as in asthmatic children and adults. A statistical meta-analysis using regression was performed for parameters of adrenal suppression in 27 studies. Results Marked adrenal suppression occurs with high doses of inhaled corticosteroid above 1.5 mg/d (0.75 mg/d for fluticasone propionate), although there is a considerable degree of interindividual susceptibility. Meta-analysis showed significantly greater potency for dose-related adrenal suppression with fluticasone compared with beclomethasone dipropionate, budesonide, or triamcinolone acetonide, whereas prednisolone and fluticasone propionate were approximately equivalent on a 10:1-mg basis. Inhaled corticosteroids in doses above 1.5 mg/d (0.75 mg/d for fluticasone propionate) may be associated with a significant reduction in bone density, although the risk for osteoporosis may be obviated by postmenopausal estrogen replacement therapy. Although medium-term growth studies showed suppressive effects with 400-µg/d beclomethasone dipropionate, there was no evidence to support any significant effects on final adult height. Long-term, high-dose inhaled corticosteroid exposure increases the risk for posterior subcapsular cataracts, and, to a much lesser degree, the risk for ocular hypertension and glaucoma. Skin bruising is most likely to occur with high-dose exposure, which correlates with the degree of adrenal suppression. Conclusions All inhaled corticosteroids exhibit dose-related systemic adverse effects, although these are less than with a comparable dose of oral corticosteroids. Meta-analysis shows that fluticasone propionate exhibits greater dose-related systemic bioactivity compared with other available inhaled corticosteroids, particularly at doses above 0.8 mg/d. The long-term systemic burden will be minimized by always trying to achieve the lowest possible maintenance dose that is associated with optimal asthmatic control and quality of life.

Nitrogen cycling in coastal marine ecosystems.

Abstract: It is generally considered that nitrogen availability is one of the major factors regulating primary production in temperate coastal marine environments. Coastal regions often receive large anthropogenic inputs of nitrogen that cause eutrophication. The impact of these nitrogen additions has a profound effect in estuaries and coastal lagoons where water exchange is limited. Such increased nutrient loading promotes the growth of phytoplankton and fast growing pelagic macroalgae while rooted plants (sea-grasses) and benthic are suppressed due to reduced light availability. This shift from benthic to pelagic primary production introduces large diurnal variations in oxygen concentrations in the water column. In addition oxygen consumption in the surface sediments increases due to the deposition of readily degradable biomass. In this review the physico-chemical and biological factors regulating nitrogen cycling in coastal marine ecosystems are considered in relation to developing effective management programmes to rehabilitate seagrass communities in lagoons currently dominated by pelagic macroalgae and/or cyanobacteria.

SUMO-1 modification activates the transcriptional response of p53

Abstract: The p53 tumour suppressor protein is regulated by ubiquitin-mediated proteasomal degradation. In normal cells p53 is constitutively ubiquitylated by the Mdm2 ubiquitin ligase. When the p53 response is activated by stress signals p53 levels rise due to inhibition of this degradative pathway. Here we show that p53 is modified by the small ubiquitin-like protein SUMO-1 at a single site, K386, in the C-terminus of the protein. Modification in vitro requires only SUMO-1, the SUMO-1 activating enzyme and ubc9. SUMO-1 and ubiquitin modification do not compete for the same lysine acceptor sites in p53. Overexpression of SUMO-1 activates the transcriptional activity of wild-type p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. The SUMO-1 modification pathway therefore acts as a potential regulator of the p53 response and may represent a novel target for the development of therapeutically useful modulators of the p53 response.

Fungal production of citric and oxalic acid: importance in metal speciation, physiology and biogeochemical processes.

Abstract: The production of organic acids by fungi has profound implications for metal speciation, physiology and biogeochemical cycles. Biosynthesis of oxalic acid from glucose occurs by hydrolysis of oxaloacetate to oxalate and acetate catalysed by cytosolic oxaloacetase, whereas on citric acid, oxalate production occurs by means of glyoxylate oxidation. Citric acid is an intermediate in the tricarboxylic acid cycle, with metals greatly influencing biosynthesis: growth limiting concentrations of Mn, Fe and Zn are important for high yields. The metal-complexing properties of these organic acids assist both essential metal and anionic (e.g. phosphate) nutrition of fungi, other microbes and plants, and determine metal speciation and mobility in the environment, including transfer between terrestrial and aquatic habitats, biocorrosion and weathering. Metal solubilization processes are also of potential for metal recovery and reclamation from contaminated solid wastes, soils and low-grade ores. Such ‘heterotrophic leaching’ can occur by several mechanisms but organic acids occupy a central position in the overall process, supplying both protons and a metal-complexing organic acid anion. Most simple metal oxalates [except those of alkali metals, Fe(III) and Al] are sparingly soluble and precipitate as crystalline or amorphous solids. Calcium oxalate is the most important manifestation of this in the environment and, in a variety of crystalline structures, is ubiquitously associated with free-living, plant symbiotic and pathogenic fungi. The main forms are the monohydrate (whewellite) and the dihydrate (weddelite) and their formation is of significance in biomineralization, since they affect nutritional heterogeneity in soil, especially Ca, P, K and Al cycling. The formation of insoluble toxic metal oxalates, e.g. of Cu, may confer tolerance and ensure survival in contaminated environments. In semiarid environments, calcium oxalate formation is important in the formation and alteration of terrestrial subsurface limestones. Oxalate also plays an important role in lignocellulose degradation and plant pathogenesis, affecting activities of key enzymes and metal oxidoreduction reactions, therefore underpinning one of the most fundamental roles of fungi in carbon cycling in the natural environment. This review discusses the physiology and chemistry of citric and oxalic acid production in fungi, the intimate association of these acids and processes with metal speciation, physiology and mobility, and their importance and involvement in key fungal-mediated processes, including lignocellulose degradation, plant pathogenesis and metal biogeochemistry.

AMEE Guide No. 14: Outcome-based education: Part 1-An introduction to outcome-based education

Abstract: SUMMARY Outcome-based education, a performance-based approach at the cutting edge of curriculum development, offers a powerful and appealing way of reforming and managing medical education.The emphasis is on the product‐ what sort of doctor will be produced‐ rather than on the educational process. In outcome-based education the educational outcomes are clearly and unambiguously speci® ed. These determine the curriculum content and its organisation, the teaching methods and strategies, the courses offered, the assessment process, the educational environment and the curriculum timetable.They also provide a framework for curriculum evaluation. A doctor is a unique combination of different kinds of abilities. A three-circle model can be used to present the learning outcomes in medical education, with the tasks to be performed by the doctor in the inner core, the approaches to the performance of the tasks in the middle area, and the growth of the individual and his or her role in the practice of medicine in the outer area. Medical schools need to prepare young doctors to practise in an increasingly complex healthcare scene with changing patient and public expectations, and increasing demands from employing authorities.Outcome-based education offers many advantages as a way of achieving this.It emphasises relevance in the curriculum and accountability, and can provide a clear and unambiguous framework for curriculum planning which has an intuitive appeal. It encourages the teacher and the student to share responsibility for learning and it can guide student assessment and course evaluation. What sort of outcomes should be covered in a curriculum, how should they be assessed and how should outcome-based education be implemented are issues that need to be addressed.

Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2.

Abstract: The PtdIns(3,4,5)P3-dependent activation of protein kinase B (PKB) by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2 respectively) is a key event in mediating the effects of signals that activate PtdIns 3-kinase. The catalytic domain of serum- and glucocorticoid-regulated protein kinase (SGK) is 54% identical with that of PKB and, although lacking the PtdIns(3,4, 5)P3-binding pleckstrin-homology domain, SGK retains the residues that are phosphorylated by PDK1 and PDK2, which are Thr256 and Ser422 in SGK. Here we show that PDK1 activates SGK in vitro by phosphorylating Thr256. We also show that, in response to insulin-like growth factor-1 (IGF-1) or hydrogen peroxide, transfected SGK is activated in 293 cells via a PtdIns 3-kinase-dependent pathway that involves the phosphorylation of Thr256 and Ser422. The activation of SGK by PDK1 in vitro is unaffected by PtdIns(3,4,5)P3, abolished by the mutation of Ser422 to Ala, and greatly potentiated by mutation of Ser422 to Asp (although this mutation does not activate SGK itself). Consistent with these findings, the Ser422Asp mutant of SGK is activated by phosphorylation (probably at Thr256) in unstimulated 293 cells, and activation is unaffected by inhibitors of PtdIns 3-kinase. Our results are consistent with a model in which activation of SGK by IGF-1 or hydrogen peroxide is initiated by a PtdIns(3,4, 5)P3-dependent activation of PDK2, which phosphorylates Ser422. This is followed by the PtdIns(3,4,5)P3-independent phosphorylation at Thr256 that activates SGK, and is catalysed by PDK1. Like PKB, SGK preferentially phosphorylates serine and threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs, and SGK and PKB inactivate glycogen synthase kinase-3 similarly in vitro and in co-transfection experiments. These findings raise the possibility that some physiological roles ascribed to PKB on the basis of the overexpression of constitutively active PKB mutants might be mediated by SGK.

The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research

Abstract: The discovery of glycosylphosphatidylinositol (GPI) membrane anchors has had a significant impact on several areas of eukaryote cell biology. Studies of the African trypanosome, which expresses a dense surface coat of GPI-anchored variant surface glycoprotein, have played important roles in establishing the general structure of GPI membrane anchors and in delineating the pathway of GPI biosynthesis. The major cell-surface molecules of related parasites are also rich in GPI-anchored glycoproteins and/or GPI-related glycophospholipids, and differences in substrate specificity between enzymes of trypanosomal and mammalian GPI biosynthesis may have potential for the development of anti-parasite therapies. Apart from providing stable membrane anchorage, GPI anchors have been implicated in the sequestration of GPI-anchored proteins into specialised membrane microdomains, known as lipid rafts, and in signal transduction events.

The role of trace metals in photosynthetic electron transport in O2-evolving organisms

Abstract: Iron is the quantitatively most important trace metal involved in thylakoid reactions of all oxygenic organisms since linear (= non-cyclic) electron flow from H2O to NADP+ involves PS II (2–3 Fe), cytochrome b6-f (5 Fe), PS I (12 Fe), and ferredoxin (2 Fe); (replaceable by metal-free flavodoxin in certain cyanobacteria and algae under iron deficiency). Cytochrome c6 (1 Fe) is the only redox catalyst linking the cytochrome b6-f complex to PS I in most algae; in many cyanobacteria and Chlorophyta cytochrome c6 and the copper-containing plastocyanin are alternatives, with the availability of iron and copper regulating their relative expression, while higher plants only have plastocyanin. Iron, copper and zinc occur in enzymes that remove active oxygen species and that are in part bound to the thylakoid membrane. These enzymes are ascorbate peroxidase (Fe) and iron-(cyanobacteria, and most al gae) and copper-zinc- (some algae; higher plants) superoxide dismutase. Iron-containing NAD(P)H-PQ oxidoreductase in thylakoids of cyanobacteria and many eukaryotes may be involved in cyclic electron transport around PS I and in chlororespiration. Manganese is second to iron in its quantitative role in the thylakoids, with four Mn (and 1 Ca) per PS II involved in O2 evolution. The roles of the transition metals in redox catalysts can in broad terms be related to their redox chemistry and to their availability to organisms at the time when the pathways evolved. The quantitative roles of these trace metals varies genotypically (e.g. the greater need for iron in thylakoid reactions of cyanobacteria and rhodophytes than in other O2-evolvers as a result of their lower PS II:PS I ratio) and phenotypically (e.g. as a result of variations in PS II:PS I ratio with the spectral quality of incident radiation).

Transcriptional Cross Talk between NF-κB and p53

Abstract: Many cellular stimuli result in the induction of both the tumor suppressor p53 and NF-κB. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of NF-κB has been shown to promote resistance to programmed cell death. These observations suggest that a regulatory mechanism must exist to integrate these opposing outcomes and coordinate this critical cellular decision-making event. Here we show that both p53 and NF-κB inhibit each other’s ability to stimulate gene expression and that this process is controlled by the relative levels of each transcription factor. Expression of either wild-type p53 or the RelA(p65) NF-κB subunit suppresses stimulation of transcription by the other factor from a reporter plasmid in vivo. Moreover, endogenous, tumor necrosis factor alpha-activated NF-κB will inhibit endogenous wild-type p53 transactivation. Following exposure to UV light, however, the converse is observed, with p53 downregulating NF-κB-mediated transcriptional activation. Both p53 and RelA(p65) interact with the transcriptional coactivator proteins p300 and CREB-binding protein (CBP), and we demonstrate that these results are consistent with competition for a limiting pool of p300/CBP complexes in vivo. These observations have many implications for regulation of the transcriptional decision-making mechanisms that govern cellular processes such as apoptosis. Furthermore, they suggest a previously unrealized mechanism through which dysregulated NF-κB can contribute to tumorigenesis and disease.

The 5-HT3B subunit is a major determinant of serotonin-receptor function.

Abstract: The neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) mediates rapid excitatory responses through ligand-gated channels (5-HT3 receptors). Recombinant expression of the only identified receptor subunit (5-HT3A) yields functional 5-HT3 receptors1. However, the conductance of these homomeric receptors (sub-picosiemens) is too small to be resolved directly, and contrasts with a robust channel conductance displayed by neuronal 5-HT3 receptors (9–17 pS)2,3,4,5,6,7. Neuronal 5-HT3 receptors also display a permeability to calcium ions and a current–voltage relationship that differ from those of homomeric receptors3,4,5,8. Here we describe a new class of 5-HT3-receptor subunit (5-HT3B). Transcripts of this subunit are co-expressed with the 5-HT3A subunit in the amygdala, caudate and hippocampus. Heteromeric assemblies of 5-HT3A and 5-HT3B subunits display a large single-channel conductance (16 pS), low permeability to calcium ions, and a current–voltage relationship which resembles that of characterized neuronal 5-HT3 channels. The heteromeric receptors also display distinctive pharmacological properties. Surprisingly, the M2 region of the 5-HT3B subunit lacks any of the structural features that are known to promote the conductance of related receptors. In addition to providing a new target for therapeutic agents, the 5-HT3B subunit will be a valuable resource for defining the molecular mechanisms of ion-channel function.

Polymorphic haplotypes of the interleukin‐10 5′ flanking region determine variable interleukin‐10 transcription and are associated with particular phenotypes of juvenile rheumatoid arthritis

Abstract: Objective. To determine the distribution of the interleukin-10 (IL-10) 5' flanking region haplotypes in children with arthritis and in controls, and to investigate the functional significance of each haplotype. Methods. Sequence-specific oligonucleotide probing was used to determine haplotype frequency. Transient transfection studies were used to investigate the transcription of reporter genes driven by each haplotype. Whole blood cultures were performed to assess IL-10 production by each genotype. Results. Patients with arthritis involving >4 joints were more likely to have a genotype with an ATA haplotype than those whose arthritis remained restricted to <4 joints. This ATA haplotype was associated with lower transcriptional activity than the GCC haplotype (P = 0.02), and the ATA/ATA genotype was associated with lower IL-10 production under lipopolysaccharide stimulation than other genotypes (P < 0.02). Conclusion. The results of this study demonstrate the functional significance of the ATA haplotype and reveal a significant association of genotypes containing this haplotype with extended oligoarthritis.

Cyanobacterial toxins, exposure routes and human health

Abstract: The production of potent toxins by bloom-, scum- and mat-forming cyanobacteria, in fresh-, brackish and marine waters, appears to be a global phenomenon. Cyanobacterial toxins can also be produced by cyanobacteria from terrestrial sources. The range and number of known cyanobacterial toxins are increasing apace as associated poisoning incidents are investigated, and increasingly powerful analytical methods are applied to complement toxicity-based studies on both natural samples and laboratory isolates of cyanobacteria. Water quality management to reduce toxic cyanobacterial mass developments, and schemes to mitigate the potential effects of cyanobacterial toxins, require an understanding of the occurrence and properties of the toxins and of the exposure routes via which the toxins present risks to health. Here, we review advances in the recognition of cyanobacterial toxins and their toxicity, and of the exposure routes with reference to human health, namely via skin contact, inhalation, haemodialysis and ...

Serine 15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2

Abstract: The p53 tumour suppressor protein is a labile transcription factor that is activated and stabilized in response to a wide range of cellular stresses, through a mechanism involving disruption of its interaction with MDM2, a negative regulatory partner. Induction of p53 by DNA damage additionally involves a series of phosphorylation and acetylation modifications, some of which are thought to regulate MDM2 binding. Here we report the effects of introducing mutations at several known or putative N‐terminal phosphorylation sites on the transactivation function of p53. These studies highlight phosphorylation of Ser15, a key phosphorylation target during the p53 activation process, as being critical for p53‐dependent transactivation. Biochemical data indicate that the mechanism by which phosphorylation of Ser15 stimulates p53‐dependent transactivation occurs through increased binding to the p300 coactivator protein. The data also indicate that Ser15‐dependent regulation of transactivation is independent of any involvement in modulating MDM2 binding, and that Ser15 phosphorylation alone is not sufficient to block the p53–MDM2 interaction.

Developmental basis of limblessness and axial patterning in snakes

Abstract: The evolution of snakes involved major changes in vertebrate body plan organization, but the developmental basis of those changes is unknown. The python axial skeleton consists of hundreds of similar vertebrae, forelimbs are absent and hindlimbs are severely reduced. Combined limb loss and trunk elongation is found in many vertebrate taxa1, suggesting that these changes may be linked by a common developmental mechanism. Here we show that Hox gene expression domains are expanded along the body axis in python embryos, and that this can account for both the absence of forelimbs and the expansion of thoracic identity in the axial skeleton. Hindlimb buds are initiated, but apical-ridge and polarizing-region signalling pathways that are normally required for limb development are not activated. Leg bud outgrowth and signalling by Sonic hedgehog in pythons can be rescued by application of fibroblast growth factor or by recombination with chick apical ridge. The failure to activate these signalling pathways during normal python development may also stem from changes in Hox gene expression that occurred early in snake evolution.

E.A.E.S. multicenter prospective randomized trial comparing two-stage vs single-stage management of patients with gallstone disease and ductal calculi

Abstract: Background: The current management of patients with gallstone disease and ductal calculi consists of endoscopic stone extraction (ESE) followed by laparoscopic cholecystectomy (LC). Following the advent of techniques of laparoscopic ductal stone clearance, an alternative single-stage laparoscopic treatment was introduced for these patients. The European Association of Endoscopic Surgery (E.A.E.S.) set up a ductal stone trial to compare the relative efficacy and outcome of these two management options.

Use of thermography for quantitative studies of spatial and temporal variation of stomatal conductance over leaf surfaces

Abstract: This paper describes a new approach to the calibration of thermal infrared measurements of leaf temperature for the estimation of stomatal conductance and illustrates its application to thermal imaging of plant leaves. The approach is based on a simple reformulation of the leaf energy balance equation that makes use of temperature measurements on reference surfaces of known conductance to water vapour. The use of reference surfaces is an alternative to the accurate measurement of all components of the leaf energy balance and is of potentially wide application in studies of stomatal behaviour. The resolution of the technique when applied to thermal images is evaluated and some results of using the approach in the laboratory for the study of stomatal behaviour in leaves of Phaseolus vulgaris L. are presented. Conductances calculated from infrared measurements were well correlated with estimates obtained using a diffusion porometer.

Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase.

Abstract: The catalytic domain of serum- and glucocorticoid-induced protein kinase (SGK) is 54% identical with protein kinase B (PKB) and, like PKB, is activated in vitro by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and in vivo in response to signals that activate phosphatidylinositol (PI) 3-kinase. Here we identify two novel isoforms of SGK, termed SGK2 and SGK3, whose catalytic domains share 80% amino acid sequence identity with each other and with SGK (renamed SGK1). Like SGK1, the mRNA encoding SGK3 is expressed in all tissues examined, but SGK2 mRNA is only present at significant levels in liver, kidney and pancreas and, at lower levels, in the brain. The levels of SGK2 mRNA in H4IIE cells and SGK3 mRNA in Rat2 fibroblasts are not increased by stimulation with serum or dexamethasone, whereas the level of SGK1 mRNA is increased greatly. SGK2 and SGK3 are activated in vitro by PDK1, albeit more slowly than SGK1, and their activation is accompanied by the phosphorylation of Thr(193) and Thr(253) respectively, the residues equivalent to the Thr in the 'activation loop' of PKB that is targeted by PDK1. The PDK1-catalysed phosphorylation and activation of SGK2 and SGK3, like SGK1, is greatly potentiated by mutating Ser(356) and Ser(419) respectively to Asp, these residues being equivalent to the C-terminal phosphorylation site of PKB. Like SGK1, SGK2 and SGK3 are activated 5-fold via a phosphorylation mechanism when cells are exposed to H(2)O(2) but, in contrast with SGK1, activation is only suppressed partially by inhibitors of PI 3-kinase. SGK2 and SGK3 are activated to a smaller extent by insulin-like growth factor-1 (2-fold) than SGK1 (5-fold). Like PKB and SGK1, SGK2 and SGK3 preferentially phosphorylate Ser and Thr residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs.