bluuebunny/crossref_metadata_2025 huggingface dataset:
Travel time distribution in a hillslope: Insight from numerical simulations
Authors: Fiori Aldo , Russo David
Published: December 2008

Solute transport in a three‐dimensional, heterogeneous hillslope is analyzed through a series of detailed, three‐dimensional numerical simulations. The investigation focuses on the transport of a pulse of a passive solute, taking into account realistic features of the relevant flow domain (i.e., the spatial heterogeneity of the soil hydraulic properties and records of the time‐dependent meteorological data and evapotranspiration). The scope of the present work is to analyze a few issues regarding the travel time probability density function (pdf) f(τ) of solute, with particular reference to the following: (1) the suitability of the time invariance assumption and the circumstance under which it may represent a valid approximation, (2) the shape of the resulting travel time pdf f(τ), and (3) the difference between f(τ) and the instantaneous unit hydrograph. It is found that in many cases of practical interest the transport of contaminants in catchments can be analyzed by assuming the time‐invariant approximation for f(τ), provided that the calendar time is replaced by a flow‐corrected time. Here f(τ) is calculated through the analysis of the breakthrough curve represented in terms of flux‐averaged concentration versus cumulate discharge. The rescaling of time with respect to the cumulated outflow takes care in an approximate way of the transient processes occurring in the porous medium. The derived f(τ) is weakly dependent on various attributes, like the level of heterogeneity, presence of evaporation or transpiration, and injection period. The main exception regards the cases in which plant transpiration is intense in the vicinity of the channel after relatively long periods of low rain. The impact of the parameters' heterogeneity on f(τ) is generally quite limited, the dispersion being ruled by the distribution of length paths within the hillslope. The derived f(τ) seems compatible with the Gamma distribution, being characterized by both fast and slow responses, with a pronounced power law early peak and an exponential‐like tail. Comparison of f(τ) with the often employed Gamma instantaneous unit hydrograph emphasizes the differences between water and solute dynamics after rainfall events.
Dopamine Release in the Human Striatum: Motor and Cognitive Tasks Revisited
Authors: Lappin Julia M , Reeves Suzanne J , Mehta Mitul A , Egerton Alice , Coulson Mark , Grasby Paul M
Published: December 2008

Striatal dopamine (DA) release has been shown during behavioural tasks, but the relative contribution of motor, reward, and cognitive components is unclear. Dopamine release was quantified using [11C]-raclopride in two studies using a triple-scan approach, comprising active task, motor control, and rest. In the first, bolus radiotracer was delivered during a sequential motor learning paradigm; in the second, a spatial planning task, bolus plus constant infusion was applied. [11C]-raclopride binding potentials (BPNDs) in striatal functional subdivisions were compared across conditions. [11C]-raclopride BPND was significantly reduced in active task compared with rest in both the sensorimotor and associative striatum in both studies, because of differences between rest and motor control conditions. In both regions, the motor control BPND fell between the rest and active task in the planning study, but the difference between motor control and active task conditions was not significant. No such changes were observed in the limbic striatum. Using rigorous methodology, this study validates earlier evidence that striatal DA release occurs during behavioural challenges. Increased DA release during movement was reliably detected in the sensorimotor and associative striatum, supporting use of the functional subdivision model in humans. No additional DA release was observed specific to the cognitive component of either task.
Reperfusion-Induced Temporary Appearance of Therapeutic Window in Penumbra after 2 h of Photothrombotic Middle Cerebral Artery Occlusion in Rats
Authors: Yao Hiroshi , Yoshii Narihiko , Akira Toshiaki , Nakahara Tatsuo
Published: December 2008

To explore the effects of reperfusion on evolution of focal ischemic injury, spontaneously hypertensive male rats were subjected to photothrombotic distal middle cerebral artery occlusion (MCAO) with or without YAG laser-induced reperfusion. The volume of fodrin breakdown zone, water content, and brain tissue levels of sodium (Na+) and potassium (K+) were measured in the ischemic core and penumbra. Reperfusion attenuated fodrin breakdown, and the volume containing fodrin breakdown product at 3 h after reperfusion (5 h after MCAO) (30±7 mm3) was significantly smaller than the 42±3 mm3of the permanent occlusion group. After 3 to 6 h of ischemia, Na+increased, and K+decreased in the ischemic core. Reperfusion after 2 h of MCA occlusion did not mitigate the ischemia-induced changes in brain tissue electrolytes and water content at 3 to 6 h of ischemia. Even in reperfusion after comparatively long periods of occlusion where brain infarction size, assessed 3 days after MCAO, was not significantly reduced by reperfusion, and the precipitating indicators of the ischemic core (Na+, K+, water content) did not improve, temporary improvement or a delay in progression of ischemic injury was discernible in the penumbra. These results indicate the possibility that treatment with reperfusion is permissive to the effects of neuroprotection.
An Analysis of Regional Microvascular Loss and Recovery following Two Grades of Fluid Percussion Trauma: A Role for Hypoxia-Inducible Factors in Traumatic Brain Injury
Authors: Park Eugene , Bell Joshua D , Siddiq Ishita P , Baker Andrew J
Published: December 2008

Secondary hypoxic/ischemic injuries, stemming from reductions in cerebral blood flow are important contributing factors in progressive neuronal dysfunction after brain trauma. A greater preclinical understanding of how brain trauma leads to secondary hypoxia/ischemia is necessary in the development of posttraumatic brain injury (TBI) therapeutics. To this end, we examined the density of microvascular coverage in the injured and contralateral cortical hemispheres using two intensities of fluid percussion trauma in rats. A silicone microangiography technique showed a significant loss in microvascular density in 2 atmosphere (atm) (16.9±3.8%) and 3 atm (15.7±1.3%) injured animals relative to sham animals (29.9±2.5%; P<0.01). RECA-1 immunohistochemistry indicated that capillary changes involved a reduction in capillary number and diameter. Reduction in microvascular density was shown to be a diffuse phenomenon occurring up to 4 mm rostral and caudal to the injury epicenter. Recovery of microvasculature occurred by 2 weeks after injury only in the 2 atm injury group. Expression of HIF1α and increased vascular endothelial growth factor expression were observed in the ipsilateral hippocampus suggesting sufficiently impaired microcirculation resulting in the expression of hypoxic-response proteins. Collectively, the results indicate diffuse and heterogeneous microvascular alterations as well as endogenous expression of neuroprotective and neovascularization pathways after TBI.
Roles of PPARΔ in the control of muscle development and metabolism
Authors: Grimaldi P.A.
Published: December 2008

PPAR δ (peroxisome proliferator-activated receptor δ)-specific agonists decrease plasma lipids and insulinaemia in obese animals. As skeletal muscle is one of the major organs for fatty acid catabolism, we have investigated the roles of the nuclear receptor in the control of muscle development and lipid metabolism, by using two approaches. We have used C2C12 myotubes in which the PPAR δ activity was altered by overexpression of either native or dominant-negative (DN) mutant forms of PPAR δ. Treatment of C2C12 cells by specific PPAR δ agonists promotes expression of genes for proteins of fatty acid catabolism and increases fatty acid oxidation. These responses were increased in C2C12-PPAR δ cells and impaired in C2C12-PPAR δDN cells. We also constructed animal models with muscle-specific expression of PPAR δ (Cre/Lox approach). The effects of muscle-specific alteration of PPAR δ activity were studied on muscle development and metabolism as well as on body fat mass. These experiments indicated that PPAR δ plays a crucial role in myofibre typing determination and regulation of muscle oxidative capabilities, and that muscle-specific overexpression of the nuclear receptor leads to reduction of adipocyte size and body fat mass. These data strongly suggest that PPAR δ controls fatty acid catabolism in muscle and that its activation by synthetic agonists could prevent or correct obesity and type 2 diabetes.
Mechanism of glucose sensing in the small intestine
Authors: Dyer J. , Vayro S. , Shirazi-Beechey S.P.
Published: December 2008

Sensing nutrients is a fundamental task for all living cells. For most eukaryotic cells glucose is a major source of energy, having significant and varied effects on cell function. Interest in identifying mechanisms by which cells sense and respond to variations in glucose concentration has increased recently. The epithelial cells lining the intestinal tract are exposed, from the luminal domain, to an environment with continuous and massive fluctuations in the levels of dietary monosaccharides. Enterocytes therefore have to sense and respond to the significant changes in the levels of luminal sugars, and regulate the expression of the intestinal glucose transporter (Na+/glucose co-transporter, SGLT1) accordingly. Our data, using a combination of in vivo and in vitro model systems, suggest that glucose in the lumen of the intestine is sensed by a glucose sensor residing on the external face of the enterocyte luminal membrane. Glucose binds to the sensor and generates an intracellular signal leading to enhancement in the expression of SGLT1. The generated signal is independent of glucose metabolism and is likely to operate via a G-protein-coupled receptor and cAMP/protein kinase A signalling cascade.
Bypassing the glucose/fatty acid cycle: AMP-activated protein kinase
Authors: Carling D. , Fryer L.G.D. , Woods A. , Daniel T. , Jarvie S.L.C. , Whitrow H.
Published: December 2008

The AMPK (AMP-activated protein kinase) cascade plays a key role in regulating energy metabolism. Conditions which cause a decrease in the ATP/AMP ratio lead to activation of AMPK. Once activated, AMPK initiates a series of responses that act to restore the energy balance of the cell. In skeletal muscle, activation of AMPK increases both glucose uptake and fatty acid oxidation, raising the possibility that AMPK can bypass the glucose/fatty acid cycle. This review focuses on the role of AMPK in the regulation of glucose and fatty acid metabolism in muscle. Recently, naturally occurring mutations within the γ isoforms have been identified which lead to altered metabolic regulation in cardiac and skeletal muscle and suggest an important role for the kinase in regulating glycogen metabolism.
Extending the glucose/fatty acid cycle: a glucose/adipose tissue cycle
Authors: Guerre-Millo M.
Published: December 2008

It is now recognized that the WAT (white adipose tissue) produces a variety of bioactive peptides, collectively termed ‘adipokines’. Alteration of WAT mass in obesity or lipoatrophy affects the production of most adipose secreted factors. Since both conditions are associated with insulin resistance, the idea has emerged that certain adipokines might influence insulin action. Among these, tumour necrosis factor α, interleukin-6 and resistin are increased in the obese state and interfere negatively with insulin-mediated processes. Conversely, leptin and adiponectin exert an insulin-sensitizing effect, at least in part by favouring tissue fatty-acid oxidation through AMP-activated kinase activation. Obesity-induced insulin resistance has been linked to leptin resistance and decreased plasma adiponectin, while administration of leptin and adiponectin normalizes plasma levels in lipoatrophic mice and reverses insulin resistance. Thiazolidinedione anti-diabetic agents increase endogenous adiponectin production in rodents and humans, supporting the idea that drugs targeting adipokines might represent a new therapeutic approach to sensitize peripheral tissues to insulin.
AZD7545, a novel inhibitor of pyruvate dehydrogenase kinase 2 (PDHK2), activates pyruvate dehydrogenase in vivo and improves blood glucose control in obese (fa/fa) Zucker rats
Authors: Mayers R.M. , Butlin R.J. , Kilgour E. , Leighton B. , Martin D. , Myatt J. , Orme J.P. , Holloway B.R.
Published: December 2008

PDH (pyruvate dehydrogenase) is a key enzyme controlling the rate of glucose oxidation, and the availability of gluconeogenic precursors. Activation of PDH in skeletal muscle and liver may increase glucose uptake and reduce glucose production. This study describes the properties of AZD7545, a novel, small-molecule inhibitor of PDHK (PDH kinase). In the presence of PDHK2, AZD7545 increased PDH activity with an EC50 value of 5.2 nM. In rat hepatocytes, the rate of pyruvate oxidation was stimulated 2-fold (EC50 105 nM). A single dose of AZD7545 to Wistar rats increased the proportion of liver PDH in its active, dephosphorylated form in a dose-related manner from 24.7 to 70.3% at 30 mg/kg; and in skeletal muscle from 21.1 to 53.3%. A single dose of 10 mg/kg also significantly elevated muscle PDH activity in obese Zucker (fa/fa) rats. Obese, insulin-resistant, Zucker rats show elevated postprandial glucose levels compared with their lean counterparts (8.7 versus 6.1 mM at 12 weeks old). AZD7545 (10 mg/kg) twice daily for 7 days markedly improved the 24-h glucose profile, by eliminating the postprandial elevation in blood glucose. These results suggest that PDHK inhibitors may be beneficial agents for improving glucose control in the treatment of type 2 diabetes.
The anti-apoptotic response of the Gq/11-coupled muscarinic receptor family
Authors: Tobin A.B. , Budd D.C.
Published: December 2008

It is now clear that G-protein-coupled receptors can regulate programmed cell death (apoptosis) through a variety of mechanisms that are dependent on cell type and receptor subtype. Here we present evidence that the Gq/11-coupled subtypes of the muscarinic receptor family (namely M1, M3 and M5-muscarinic receptor subtypes) are able to protect against apoptotic cell death. In particular we demonstrate that the C-terminal tail of the M3-muscarinic receptor is an essential structural element for signalling to the anti-apoptotic pathway. Removal of the distal portion of the C-terminal tail results in a receptor that is coupled normally to the Gq/11/phospholipase C pathway and the mitogen-activated protein kinase pathway, but is unable to couple to the anti-apoptotic pathway. Furthermore, a poly-basic region conserved within the C-terminal tail of the Gq/11-coupled muscarinic receptor subtypes appears to be the structural determinant of coupling to the anti-apoptotic pathway.
The role of ERK2 docking and phosphorylation of PDE4 cAMP phosphodiesterase isoforms in mediating cross-talk between the cAMP and ERK signalling pathways
Authors: Houslay M.D. , Baillie G.S.
Published: December 2008

PDE4 cAMP phosphodiesterases are widely expressed enzymes that serve as major regulators of cAMP signalling in cells. They provide targets for therapeutics having anti-inflammatory and cognitive-enhancing properties. ERK2 (extracellular-signal-regulated kinase 2) interacts with the PDE4 catalytic unit by binding to a KIM (kinase interaction motif) docking site located on an exposed β-hairpin loop and an FQF (Phe-Gln-Phe) specificity site located on an exposed α-helix. These flank a site that allows phosphorylation by ERK, the functional outcome of which is orchestrated by the N-terminal UCR1/2 (upstream conserved region 1 and 2) modules. The three classes of PDE4 isoforms differ in these regulatory modules, allowing phosphorylation by ERK to lead to either inhibition or activation. ERK inhibition of long isoforms is regulated by a unique feedback control whereby elevated cAMP levels cause PKA (protein kinase A) to phosphorylate UCR1 and ablate the inhibitory action of ERK. PDE4 isoforms can also be found in complex with β-arrestins where they provide a novel part of the cellular desensitization mechanism to receptor-mediated cAMP signalling. Stimulation of the β2-adrenoceptor recruits β-arrestins with bound PDE4, delivering an enzyme capable of degrading cAMP at its site of synthesis at the plasma membrane. Use of dominant negative PDE4 isoforms identifies that a major role of recruited PDE4 is to regulate plasma membrane PKA activity involved in phosphorylating the β2-adrenoceptor. Recruited PDE4 thus desensitizes the ability of the β2-adrenoceptor to activate ERK via Gi.
Protease-activated receptor 2: activation, signalling and function
Authors: Cottrell G.S. , Amadesi S. , Schmidlin F. , Bunnett N.
Published: December 2008

PARs (protease-activated receptors) are a family of four G-protein-coupled receptors for proteases from the circulation, inflammatory cells and epithelial tissues. This report focuses on PAR2, which plays an important role in inflammation and pain. Pancreatic (trypsin I and II) and extrapancreatic (trypsin IV) trypsins, mast cell tryptase and coagulation factors VIIa and Xa cleave and activate PAR2. Proteases cleave PAR2 to expose a tethered ligand that binds to the cleaved receptor. Despite this irreversible activation, PAR2 signalling is attenuated by β-arrestin-mediated desensitization and endocytosis, and by lysosomal targeting and degradation, which requires ubiquitination of PAR2. β-Arrestins also act as scaffolds for the assembly of multi-protein signalling complexes that determine the location and function of activated mitogen-activated protein kinases. Observations of PAR2-deficient mice support a role for PAR2 in inflammation, and many of the effects of PAR2 activators promote inflammation. Inflammation is mediated in part by activation of PAR2 in the peripheral nervous system, which results in neurogenic inflammation and hyperalgesia.
How does the translocon differentiate between hydrophobic sequences that form part of either a GPI (glycosylphosphatidylinositol)-anchor signal or a stop transfer sequence?
Authors: Dalley J.A. , Bulleid N.J.
Published: December 2008

Newly synthesized proteins entering the eukaryotic secretory pathway may be attached to the lipid membrane by essentially one of two mechanisms. They may either contain a hydrophobic stop transfer sequence that directs their integration into the bilayer with the consequence that the polypeptide spans the membrane either one or several times, or alternatively the polypeptide chain may be modified by the covalent addition of a lipid anchor resulting in the attachment of the protein to the membrane via the lipid moiety. The major pathway for the covalent addition of a lipid anchor involves the post-translational attachment of GPI (glycosylphosphatidylinositol) to the C-terminus. Proteins modified in this way contain a specific signal that is recognized by the GPI-anchor processing machinery. Hence both the integration of protein directly into the lipid bilayer and the addition of GPI anchors require the presence of sequences within the polypeptide chain to target the proteins to these pathways. This article will describe the main characteristics of these signals and their similarities and will discuss how the translocon may play a crucial role in their recognition.
Insulin signalling, exercise and cellular integrity
Authors: Kirwan J.P. , del Aguila L.F.
Published: December 2008

Although the effects of exercise on insulin sensitivity are generally positive, eccentric exercise presents a paradox because it induces a transient state of insulin resistance that persists for up to 48 h after the exercise bout. Excessive eccentric contractions, such as prolonged downhill running, or marathon running, causes muscle damage and disruption of the integrity of the cell. Down-regulation of insulin receptor tyrosine phosphorylation and subsequent steps in the insulin signalling pathway, including insulin receptor substrate-1 (IRS-1)-associated phosphoinositide 3-kinase (PI3K), Akt kinase serine phosphorylation and activity and glucose transporter (GLUT-4) protein content, are evident in skeletal muscle after eccentric exercise. Furthermore, increased tumour necrosis factor α (TNF-α) secretion from monocytes is associated with the decrease in PI3K activity after this type of exercise. Recent studies have shown that TNF-α can increase IRS-1 serine/threonine phosphorylation, which impairs IRS-1 docking to the insulin receptor, and this inhibits insulin signalling. Thus a unifying hypothesis to explain insulin resistance after eccentric exercise may include inflammation arising from the disruption of muscle-cell integrity, leading to an acute-phase response that includes TNF-α, with the latter inhibiting insulin signalling and subsequent metabolic events. In contrast, exercise training increases insulin signalling and GLUT-4 expression, decreases TNF-α expression in skeletal muscle, and is associated with enhanced insulin sensitivity. These observations highlight the complexity of the cellular and molecular adaptations to exercise. Understanding these adaptations is essential in order to establish a sound theoretical basis for recommending exercise as a therapeutic intervention for insulin resistance and type 2 diabetes.
The renin–angiotensin system and physical performance
Authors: Payne J. , Montgomery H.
Published: December 2008

Many of us recognize that some individuals seem ‘gifted’ in sporting ability. We may also have noted the association of such elite performance with past parental success, recognizing intuitively the role of inherited traits. With the expansion of molecular biology and associated technologies, we now find ourselves better able to explore these genetic influences. This article examines the role of the renin–angiotensin system in regulating physical performance, based on data arising from candidate gene-association studies. In particular, the association of angiotensin-converting enzyme genotype with performance-related phenotypes will be addressed. Finally, we will briefly discuss the applicability of this data to disease states such as heart failure.
Prokaryotic Cu,Zn superoxide dismutases
Authors: Desideri A. , Falconi M.
Published: December 2008

The Cu,ZnSODs (Cu,Zn superoxide dismutases) comprise a class of ubiquitous metalloenzymes that catalyse the dismutation of the superoxide radical anion into oxygen and hydrogen peroxide. The dismutation reaction involves two successive encounters of the superoxide anion with a catalytic copper centre hosted by the enzyme at the dead end of a narrow protein channel. Cu,ZnSOD is found in all eukaryotic species as a homodimeric enzyme of approx. 2×16 kDa. Cu,ZnSODs are also widely distributed in the prokaryotic phyla, and are found in the periplasmic space of bacterial pathogens, where they are likely to play a defensive role against extracellular oxidative stress, particularly with respect to phagocytic processes. The crystallographic structures of prokaryotic Cu,ZnSODs have shown that the core structural organization of the enzyme is based on a flattened antiparallel β-barrel, composed of eight strands arranged in a Greek-key topological order, which usually forms a dimeric structure through the interaction of residues provided by β-strands 4f and 5e with the 2,3 and 7,8 loops. The interface residues are arranged to form a closed ring, which surrounds a central cavity filled by water molecules that are arranged differently depending on the species. The interface variability of residues and solvent structure observed in bacterial enzymes permits fine tuning of the subunit association that results in the presence of monomeric and dimeric species, and confers a particular protein flexibility that gives rise to long-range communications between different region of the enzyme.
Role of prokaryotic Cu,Zn superoxide dismutase in pathogenesis
Authors: Battistoni A.
Published: December 2008

Several bacterial pathogens possess sodC genes that encode periplasmic or membrane-associated Cu,Zn superoxide dismutases. Since professional phagocytes generate large amounts of reactive oxygen species to control the growth of invading micro-organisms, Cu,Zn superoxide dismutase might protect infectious bacteria from oxy-radical damage and facilitate their survival within the host. This idea has gained support from studies showing that sodC-null mutants of different bacteria are less virulent than their parental wild-type strains, and from the discovery that, despite apparent dispensability for growth under laboratory conditions, various pathogens (including several highly virulent Salmonella strains) possess multiple copies of sodC. Our studies indicate that Cu,Zn superoxide dismutase effectively protects bacteria from phagocytic killing, and that the role in infection of the redundant sodC genes may vary in distinct Salmonella enterica serovars. More unexpectedly, we have found that Cu,Zn superoxide dismutase also modulates bacterial survival within epithelial cells, where bacterial killing appears to be mediated by an NAD(P)H oxidase resembling the enzyme complex typical of phagocytes. Finally, a striking feature of Cu,Zn superoxide dismutases from bacterial pathogens is their apparent ability to exploit the structural versatility of the enzyme to modulate its function. In fact, several enzyme variants exhibit unique properties that may lead to the acquisition of novel specialized functions distinct from superoxide dismutation.
Fructosamine 3-kinase, an enzyme involved in protein deglycation
Authors: Delpierre G. , Van Schaftingen E.
Published: December 2008

The in vivo formation of fructosamines following non-enzymatic reaction of proteins with glucose (i.e. glycation) was first described almost 30 years ago. Until recently, the only known fate of fructosamines in mammalian cells was their spontaneous conversion into advanced glycation end products. The identification in human erythrocytes of a new enzyme, fructosamine 3-kinase, disclosed the existence of a so-far unsuspected intracellular metabolism of these compounds. Fructosamine 3-kinase phosphorylates with high affinity both low-molecular-mass and protein-bound fructosamines on the third carbon of their deoxyfructose moiety, leading to the formation of fructosamine 3-phosphates. The latter are unstable and spontaneously decompose into inorganic phosphate and 3-deoxyglucosone, with concomitant regeneration of the unglycated amine. The presence of proteins related to fructosamine 3-kinase in many prokaryotic and eukaryotic genomes suggests that this ‘deglycation’ process is not restricted to mammals.
α-Oxoaldehyde metabolism and diabetic complications
Authors: Beisswenger P.J. , Howell S.K. , Nelson R.G. , Mauer M. , Szwergold B.S.
Published: December 2008

The factors responsible for variable susceptibility to diabetic nephropathy are not clear. According to the non-enzymatic glycation hypothesis, diabetes-related tissue damage occurs due to a complex mixture of toxic products, including α-oxoaldehydes, which are inherently toxic as well as serving as presursors for advanced glycation end-products. Protective mechanisms exist to control this unavoidable glycation, and these are determined by genetic or environmental factors that can regulate the concentrations of the reactive sugars or end-products. In diabetes these protective mechanisms become more important, since glycation stress increases, and less efficient defence systems against this stress could lead to diabetic complications. Some of these enzymatic control mechanisms, including those that regulate α-oxoaldehydes, have been identified. We have observed significant increases in production of the α-oxoaldehydes methylglyoxal and 3-deoxyglucosone in three human populations with biopsy-proven progression of nephropathy. The increase in methylglyoxal could be secondary to defects in downstream glycolytic enzymes (such as glyceraldehyde-3-phosphate dehydrogenase) that regulate its production, or in detoxification mechanisms such as glyoxalase. Other mechanisms, however, appear to be responsible for the observed increase in 3-deoxyglucosone levels. We present results of our studies on the mechanisms responsible for variable production of α-oxoaldehydes by measuring the activity and characteristics of these enzymes in cells from complication-prone and -resistant diabetic patients. New therapeutic interventions designed to control these endogenous mechanisms could potentially enhance protection against excessive glycation and prevent or reverse complications of long-term diabetes.
Role of genetic variability in systems of receptor-mediated and enzymatic defence against glycation in the long-term consequences of diabetes mellitus
Authors: Kaňková K.
Published: December 2008

Diabetes mellitus is the most common metabolic disease, and has late complications that are due to chronic hyperglycaemia. Altered carbohydrate and lipid metabolism together with impaired detoxification of carbonyl substrates and impaired trapping of oxygen radicals are responsible for cell damage in diabetes. Variable functional capacity of detoxifying systems could contribute to differing susceptibility to the development of complications. Identification of genetic variants responsible for modulating relevant intermediate phenotypes in diabetics could help to define individual risk profiles and to modify therapeutic strategy accordingly.
Glycation in food and metabolic transit of dietary AGEs (advanced glycation end-products): studies on the urinary excretion of pyrraline
Authors: Foerster A. , Henle T.
Published: December 2008

Pyrraline [∊-(2´-formyl-5´-hydroxymethyl-pyrrolyl)-l-norleucin] belongs to the group of AGEs (advanced glycation end-products) formed in the final stage of the Maillard reaction in foods and in vivo. As it is generally accepted that AGEs are pathophysiologically relevant in aging and in diseases such as diabetes and uraemia, physiological consequences resulting from the ingestion of dietary AGEs are discussed, but balance studies for well defined AGEs are still lacking. The aim of our study was to investigate the influence of nutrition on the urinary excretion of pyrraline. After the first day without dietary restrictions, seven healthy volunteers were asked, starting on the morning of day 2, to ingest a diet virtually free of Maillard compounds (i.e. no cooked or roasted foods, no bakery products, no coffee, etc.). Dietary control was stopped on the morning of day 5. We collected 24 h urine samples for these 5 days, which were analysed for free pyrraline by reverse-phase HPLC with UV detection at 297 nm. We found that urinary excretion of free pyrraline was directly affected by the composition of the diet, decreasing from 4.8±1.1 mg/day on day 1 to levels of 1.6, 0.4 and 0.3 mg/day on days 2, 3 and 4 respectively, followed by a significant increase to 3.2±1.4 mg/day on the 5th day. The results of this work prove, for the first time, that urinary excretion of pyrraline is strongly dependent on its dietary intake. Thus the influence of nutrition should be taken into consideration in studies directed to the physiological role of glycation compounds.
Removal of advanced glycation end products in clinical renal failure by peritoneal dialysis and haemodialysis
Authors: Agalou S. , Ahmed N. , Dawnay A. , Thornalley P.J.
Published: December 2008

AGEs (advanced glycation end products) accumulate markedly in the plasma of human subjects with renal failure. We investigated the efficiency of removal of AGEs from the circulation by PD (peritoneal dialysis) and HD (haemodialysis) therapy. Free AGEs were measured by LC-MS/MS in blood plasma before dialysis, in dialysis fluid effusate after a 2–12 h dwell time in the peritoneal cavity of PD subjects, and in the HD dialysate before and after HD therapy. In clinical uraemia, the concentrations of free AGEs in blood plasma were increased up to 50-fold. For example, levels of MG-H1 (methylglyoxal-derived hydroimidazolone) were: normal controls, 110±46 nM; PD subjects, 1876±676 (P<0.01); HD subjects, 5496±1138 nM (P<0.001). In PD subjects, the AGE concentration in the effusate increased with increasing dwell time, reaching a maximum at a concentration higher than that in plasma for some AGEs at 4–12 h. This may reflect AGE formation in the peritoneal cavity. In HD, AGE concentrations in HD fluid were decreased markedly from the start to the end of a dialysis session, except that levels of the methylglyoxal-derived AGEs N∊-(1-carboxyethyl)lysine and MG-H1, and of pentosidine, remained 5-fold higher than control levels. Inadequate clearance of free AGEs may be linked to the increased risk of cardiovascular disease in patients with renal failure.
Role of methylglyoxal adducts in the development of vascular complications in diabetes mellitus
Authors: Bourajjaj M. , Stehouwer C.D.A. , van Hinsbergh V.W.M. , Schalkwijk C.G.
Published: December 2008

Various theories have been proposed to explain the hyperglycaemia-induced pathogenesis of vascular complications of diabetes, including detrimental effects of AGEs (advanced glycation end products) on vascular tissues. Increased formation of the very reactive dicarbonyl compound MGO (methylglyoxal), one of the side-products of glycolysis, and MGO-derived AGEs seem to be implicated in the development of diabetic vascular complications. Although the exact role of MGO and MGO adducts in the development of vascular complications is unknown, receptor-mediated activation of vascular cells by the MGO–arginine adduct hydroimidazolone, as well as intracellular modifications of protein by MGO, seem to be involved. The aim of this mini-review is to assess to what extent MGO is related to vascular complications in diabetes.
Anti-glycation defences in yeast
Authors: Ponces Freire A. , Ferreira A. , Gomes R. , Cordeiro C.
Published: December 2008

Saccharomyces cerevisiae is an outstanding cellular model for metabolic studies in glycation. Due to its high glycolytic activity, it produces methylglyoxal, a highly reactive intracellular glycation agent, at a rate of approx. 0.1% of the glycolytic flux. We investigated methylglyoxal metabolism in Saccharomyces cerevisiae cells, using haploid null mutants. Growth studies showed that the most sensitive strains to 2-oxoaldehydes were the null mutants for GSH1 and GLO1, coding for glutathione synthase I and glyoxalase I respectively. The GRE3 null mutant, lacking aldose reductase activity, is as sensitive as the control strain. Kinetic modelling and computer simulation of this type of experiment were also performed, and we concluded that the most important parameters for controlling the intracellular concentration of methylglyoxal are the activity of glyoxalase I and the GSH concentration. Moreover, our model predicts an intracellular steady-state concentration of methylglyoxal of approx. 2 μM. Our results show that the glyoxalase pathway is the main detoxification pathway for 2-oxoaldehydes in yeast, and is likely to be the key enzymatic anti-glycation agent in these cells.
Role of lipids in chemical modification of proteins and development of complications in diabetes
Authors: Januszewski A.S. , Alderson N.L. , Metz T.O. , Thorpe S.R. , Baynes J.W.
Published: December 2008

Hyperglycaemia is the major risk factor for the development of complications in both Type I and Type II diabetes; however, there is growing evidence from several clinical trials that dyslipidaemia, including hypertriglyceridaemia, is a significant and independent risk factor for diabetic complications. In this paper, we propose that chemical modification of proteins by lipids may be a underlying pathogenic mechanism linking dyslipidaemia to diabetic complications. Thus the major AGEs (advanced glycation end-products) in tissues, such as carboxymethyl-lysine, carboxyethyl-lysine and hydroimidazolones, may, in fact, be ALEs (advanced lipoxidation end-products), derived from lipids. Increased lipid peroxidation and accelerated ALE formation, possibly catalysed by hyperglycaemia and oxidative stress, may be the mechanistic link between dyslipidaemia and diabetic complications. If correct, this proposal would suggest that inhibition or reversal of glycation, which is a central theme of this symposium, may not be sufficient for protection against diabetic complications.
Accumulation of fructosyl-lysine and advanced glycation end products in the kidney, retina and peripheral nerve of streptozotocin-induced diabetic rats
Authors: Karachalias N. , Babaei-Jadidi R. , Ahmed N. , Thornalley P.J.
Published: December 2008

The accumulation of AGEs (advanced glycation end products) in diabetes mellitus has been implicated in the biochemical dysfunction associated with the chronic development of microvascular complications of diabetes – nephropathy, retinopathy and peripheral neuropathy. We investigated the concentrations of fructosyl-lysine and AGE residues in protein extracts of renal glomeruli, retina, peripheral nerve and plasma protein of streptozotocin-induced diabetic rats and normal healthy controls. Glycation adducts were determined by LC with tandem MS detection. In diabetic rats, the fructosyl-lysine concentration was increased markedly in glomeruli, retina, sciatic nerve and plasma protein. The concentrations of N∊-carboxymethyl-lysine and N∊-carboxyethyl-lysine were increased in glomeruli, sciatic nerve and plasma protein, and N∊-carboxymethyl-lysine also in the retina. Hydroimidazolone AGEs derived from glyoxal, methylglyoxal and 3-deoxylglucosone were major AGEs quantitatively. They were increased in the retina, nerve, glomeruli and plasma protein. AGE accumulation in renal glomeruli, retina, peripheral nerve and plasma proteins is consistent with a role for AGEs in the development of nephropathy, retinopathy and peripheral neuropathy in diabetes. High-dose therapy with thiamine and Benfotiamine suppressed the accumulation of AGEs, and is a novel approach to preventing the development of diabetic complications.
Enzymatic deglycation – a new paradigm or an epiphenomenon?
Authors: Szwergold B.S. , Beisswenger P.J.
Published: December 2008

Following the discovery of FN3K (fructosamine 3-kinase), and more recently of FN3KRP (FN3K-related protein), research in our laboratory has been focused on testing the enzymatic deglycation hypothesis and investigating the roles of FN3K and FN3KRP. Thus far, using human erythrocytes as a model system, we have obtained the following evidence of enzymatic deglycation: (a) production of GHb (glycated haemoglobin) by d-glucose in intact erythrocytes is 5-fold lower than in haemolysates; (b) glycation of GHb by d-glucose in intact erythrocytes is 5-fold lower than that by l-glucose; (c) inhibition of ATP production in erythrocytes leads to an acceleration in the rate of GHb production by d-glucose; and (d) inhibition of FN3K in erythrocytes by a competitive inhibitor increases the accumulation of GHb. In spite of these data supporting the enzymatic deglycation hypothesis, some outstanding issues remain. These include, among others, the fact that while the apparent deglycation mechanism does not operate on l-glucose, semi-purified FN3K appears to be able to use both d- and l-fructosamines as substrates. Moreover, analysis of the fructoselysine 3-phosphate content of haemoglobin from diabetic subjects suggests that, in addition to FN3K, another deglycating mechanism may be operative in human erythrocytes. Elucidation of these issues is a challenge in the evolving field of deglycation research. Most important, however, is the question of whether enzymatic deglycation is truly an important defence mechanism or merely an epiphenomenon.
Modulation of in vivo 3-deoxyglucosone levels
Authors: Brown T.R. , Su B. , Brown K.A. , Schwartz M.A. , Tobia A.M. , Kappler F.
Published: December 2008

Fructoselysine 3-phosphate is synthesized in vivo by the recently discovered fructoseamine-3-kinase (F3K) from fructoselysine and ATP and decomposes to lysine, Pi and 3-deoxyglucosone (3DG). This pathway appears to dominate 3DG production in vivo, making it possible to modulate 3DG levels by stimulating or inhibiting the reaction. Present inhibitors are non-reacting substrate analogues with relatively high Ki values and can inhibit F3K sufficiently in vivo to reduce 3DG in diabetic rat plasma by approx. 50%. Stimulation of the F3K pathway by feeding glycated casein causes an increase of 10–20-fold in plasma levels of 3DG and 3-fold in kidney tubules. Consequences of this increase were studied in two systems: the Eker rat, a model of susceptible kidney tubules; and birth rates in two rat strains. In both cases substantial pathological effects were observed. In the Eker rats, an approx. 3-fold increase in kidney lesions was observed (P<0.00001). In both Fischer 344 and Sprague–Dawley rats, birth rates were reduced by 56% (P<0.0001) and 12% (P<0.015) respectively. These results suggest that inhibition of F3K is a promising new therapeutic target for diabetic complications, as well as other 3DG-dependent pathologies.
Application of monoclonal antibody libraries for the measurement of glycation adducts
Authors: Nagai R. , Horiuchi S.
Published: December 2008

Immunological approaches have been used to demonstrate the presence of advanced glycation end-products (AGEs) in several human and experimental animal tissues. We previously prepared polyclonal and monoclonal anti-AGE antibodies by immunizing AGE-modified proteins such as BSA and RNase. Although these antibodies contributed to demonstrate the presence of AGE-modified protein in vivo, the epitope structure of these antibodies had not been identified. We subsequently prepared several antibodies against AGE structures such as pentosidine, pyrraline, 3-deoxyglucosone imidazolone and N∊-(carboxyethyl)lysine by immunizing single AGE structures. These structure-specific antibodies have greatly helped broaden our understanding of AGE structures in aging and age-enhanced disease process. Monoclonal anti-AGE antibody is also used for the identification of major AGE structures in some pathological tissues, such as human atherosclerosis lesions. Based on the strategy, we successfully identified a novel AGE structure named glycolaldehyde-pyridine, which is the major antigenic AGE derived from glycolaldehyde. Therefore a monoclonal antibody library for AGE structures has served an important role in the elucidation of the biological significance of AGE.
Extracting meaning from microarray data
Authors: Curtis R.K. , Brand M.D.
Published: December 2008

Gene expression is complex: many mRNAs change in abundance in response to a new condition. But while some of these expression changes may be direct, many may be downstream, indirect effects. One of the major problems of microarray data analysis is distinguishing between these changes. Some of the most common methods of analysis are discussed, in the context of their ability to distinguish between direct and indirect expression changes. The application of modular control analysis to microarray data in order to partition and quantify the importance of mRNA clusters in mediating responses is described.
On the structure of protein–protein interaction networks
Authors: Thomas A. , Cannings R. , Monk N.A.M. , Cannings C.
Published: December 2008

We present a simple model for the underlying structure of protein–protein pairwise interaction graphs that is based on the way in which proteins attach to each other in experiments such as yeast two-hybrid assays. We show that data on the interactions of human proteins lend support to this model. The frequency of the number of connections per protein under this model does not follow a power law, in contrast to the reported behaviour of data from large-scale yeast two-hybrid screens of yeast protein–protein interactions. Sampling sub-graphs from the underlying graphs generated with our model, in a way analogous to the sampling performed in large-scale yeast two-hybrid searches, gives degree distributions that differ subtly from the power law and that fit the observed data better than the power law itself. Our results show that the observation of approximate power law behaviour in a sampled sub-graph does not imply that the underlying graph follows a power law.
Supervised classification for gene network reconstruction
Authors: Soinov L.A.
Published: December 2008

One of the central problems of functional genomics is revealing gene expression networks – the relationships between genes that reflect observations of how the expression level of each gene affects those of others. Microarray data are currently a major source of information about the interplay of biochemical network participants in living cells. Various mathematical techniques, such as differential equations, Bayesian and Boolean models and several statistical methods, have been applied to expression data in attempts to extract the underlying knowledge. Unsupervised clustering methods are often considered as the necessary first step in visualization and analysis of the expression data. As for supervised classification, the problem mainly addressed so far has been how to find discriminative genes separating various samples or experimental conditions. Numerous methods have been applied to identify genes that help to predict treatment outcome or to confirm a diagnosis, as well as to identify primary elements of gene regulatory circuits. However, less attention has been devoted to using supervised learning to uncover relationships between genes and/or their products. To start filling this gap a machine-learning approach for gene networks reconstruction is described here. This approach is based on building classifiers – functions, which determine the state of a gene's transcription machinery through expression levels of other genes. The method can be applied to various cases where relationships between gene expression levels could be expected.
Petri Net representations in systems biology
Authors: Pinney J.W. , Westhead D.R. , McConkey G.A.
Published: December 2008

The mathematical structures known as Petri Nets have recently become the focus of much research effort in both the structural and quantitative analysis of all kinds of biological networks. This review provides a very brief summary of these interesting new research directions.
The growth of AA graphite on (111) diamond
Authors: Lee Jae-Kap , Lee Seung-Cheol , Ahn Jae-Pyoung , Kim Soo-Chul , Wilson John I. B. , John Phillip
Published: December 2008

Stacked AA graphite has been synthesized using a high-density dc plasma in hydrogen-methane mixtures. Graphene layers have been grown epitaxially with 2-1 registration between the AA graphitic edges and the (111) surface of diamond. In addition, a new graphite crystal structure containing AA′ graphene layers, where alternate planes are translated by half the hexagon width, is formed by 1-1 registry. The resulting interplanar distances of the AA graphite at the interface range from 2.20Å for the 1-1 registration to 4.40Å for the 2-1 registration and have been measured directly by high-resolution transmission electron microscopy (TEM). The appearance of the characteristic d-spacings, 3.55, 2.15, 1.80, 1.75 (not fully resolved), and 1.25Å in the selective area diffraction patterns from the TEM, are consistent with reflections from the (001), (100), (102), (002), and (110) planes of the AA graphite. Simulation of the diffraction patterns, employing the structural factors of graphene, confirms the existence of AA graphite.