NADPH oxidase: an enzyme for multicellularity?

Multicellularity has evolved several times during the evolution of eukaryotes. One evolutionary pressure that permits multicellularity relates to the division of work, where one group of cells functions as nutrient providers and the other in specialized roles such as defence or reproduction. This requires signalling systems to ensure harmonious development of multicellular structures. Here, we show that NADPH oxidases are specifically present in organisms that differentiate multicellular structures during their life cycle and are absent from unicellular life forms. The biochemical properties of these enzymes make them ideal candidates for a role in intercellular signalling.     

Chemical modification of coenzyme B12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal-P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH₄. ?-N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH₄-reduced enzyme-pyridoxal-P complex. The relationship of inactivation vs pyridoxal-P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal-P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal-P results from irreversible dissociation of the enzyme into subunits upon pyridoxal-P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal-P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).     

SNOR and wheeze: the asthma enzyme?

Conventionally, asthma is defined as involving both airway inflammation and airway smooth muscle hyper-responsiveness. However, Que and coworkers have recently uncoupled these concepts, showing that mice lacking an S-nitrosothiol reductase have allergen-induced airway inflammation but do not have airway hyper-responsiveness. These data are consistent with recent clinical evidence that: (i) S-nitrosothiol signaling is abnormal in human asthma, (ii) nitric oxide in exhaled air might be only a biomarker for the metabolism of more physiologically relevant nitrogen oxides and (iii) the biochemical response to airway inflammation is central to asthma pathophysiology.     

Histone acetyltransferase 1: More than just an enzyme?

Histone acetyltransferase 1 (HAT1) is an enzyme that is likely to be responsible for the acetylation that occurs on lysines 5 and 12 of the NH(2)-terminal tail of newly synthesized histone H4. Initial studies suggested that, despite its evolutionary conservation, this modification of new histone H4 played only a minor role in chromatin assembly. However, a number of recent studies have brought into focus the important role of both this modification and HAT1 in histone dynamics. Surprisingly, the function of HAT1 in chromatin assembly may extend beyond just its catalytic activity to include its role as a major histone binding protein. These results are incorporated into a model for the function of HAT1 in histone deposition and chromatin assembly. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.     

Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide:N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994)J Biol Chem 269, 17611–18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g.K _m=114 µm for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase; the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man1 3(Man1 6)Man, but not by mannose and -methyl-d-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition proteinin vivo. Such dual properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases — PNGase A and PNGase F, respectively.Abbreviations GLC gas liquid chromatography - GlcNAc-Asn 2-acetamido-1--(l-aspartamido)-1,2-dideoxy-d-glucose - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose; triomannose, Man1 3(Man1 6)Man; - MES 2-(N-morphorino)ethanesulfonic acid - NeuAc N-acetyl-neuraminic acid - PNGase peptide:N ⁴-(N-accetyl-glucosaminyl)asparagine amidase (peptide:N-glycanase,EC 3.5.1.52) - PNP p-nitrophenyl     

Mitochondrial dysfunction and Down's syndrome: is there a role for coenzyme Q(10) ?

Structural changes and abnormal function of mitochondria have been documented in Down's syndrome (DS) cells, patients, and animal models. DS cells in culture exhibit a wide array of functional mitochondrial abnormalities including reduced mitochondrial membrane potential, reduced ATP production, and decreased oxido-reductase activity. New research has also brought to central stage the prominent role of oxidative stress in this condition. This review focuses on recent advances in the field with a particular emphasis on novel translational approaches involving the utilization of coenzyme Q(10) (CoQ(10) ) to treat a variety of clinical phenotypes associated with DS that are linked to increased oxidative stress and energy deficits. CoQ(10) has already provided promising results in several different conditions associated with altered energy metabolism and oxidative stress in the CNS. Two studies conducted in Ancona investigated the effect of CoQ(10) treatment on DNA damage in DS patients. Although the effect of CoQ(10) was evidenced only at single cell level, the treatment affected the distribution of cells according to their content in oxidized bases. In fact, it produced a strong negative correlation linking cellular CoQ(10) content and the amount of oxidized purines. Results suggest that the effect of CoQ(10) treatment in DS not only reflects antioxidant efficacy, but likely modulates DNA repair mechanisms.     

A role for reduced coenzyme Q in atherosclerosis?

Substantial evidence implicates oxidative modification of low density lipoprotein (LDL) as an important event contributing to atherogenesis. As a result, the elucidation of the molecular mechanisms by which LDL is oxidized and how such oxidation is prevented by antioxidants has been a significant research focus. Studies on the antioxidation of LDL lipids have focused primarily on alpha-tocopherol (alpha-TOH), biologically and chemically the most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. In addition to alpha-TOH, plasma LDL also contains low levels of ubiquinol-10 (CoQ10H2; the reduced form of coenzyme Q10). Recent studies have shown that in oxidizing plasma lipoproteins alpha-TOH can exhibit anti- or pro-oxidant activities for the lipoprotein's lipids exposed to a vast array of oxidants. This article reviews the molecular action of alpha-TOH in LDL undergoing "mild" radical-initiated lipid peroxidation, and discusses how small levels of CoQ10H2 can represent an efficient antioxidant defence for lipoprotein lipids. We also comment on the levels alpha-TOH, CoQ10H2 and lipid oxidation products in the intima of patients with coronary artery disease and report on preliminary studies examining the effect of coenzyme Q10 supplementation on atherogenesis in apolipoprotein E knockout mice.     

Is leaf ADP-glucose pyrophosphorylase an allosteric enzyme?

Barley leaf ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch synthesis in the chloroplast stroma, was analysed, in both directions of the reaction, with respect to details of its regulation by 3-phosphoglycerate (PGA) and inorganic phosphate (Pi) which serve as activator and inhibitor, respectively. AGPase was found to catalyse a close-to-equilibrium reaction, with the K(eq) value of approximately 0.5, i.e. slightly favouring the pyrophosphorolytic direction. When the enzyme was analysed by substrate kinetics, PGA acted either as a linear (hyperbolic response) 'non-competitive' activator (forward reaction) or a linear near-'competitive' activator (reverse reaction). When the activation and inhibition patterns with PGA and Pi, respectively, were studied in detail by Dixon plots, the response curves to effectors also followed hyperbolic kinetics, with the experimentally determined K(a) and K(i) values on the order of micromolar. The results suggest that the regulation of AGPase proceeds via a non-cooperative mechanism, where neither of the effectors, when considered separately, induces any allosteric response. The evidence, discussed in terms of an overall kinetic mechanism/regulation of leaf AGPase, prompts caution in classifying the protein as an 'allosteric enzyme'.     

How does an enzyme recognize CO2?

Phosphoenolpyruvate carboxykinase (PCK) reversibly catalyzes the carboxylation of phosphoenolpyruvate to oxaloacetate. Carbon dioxide, and not bicarbonate ion, is the substrate utilized. Assays of the carboxylation reaction show that initial velocities are 7.6-fold higher when CO(2) is used instead of HCO(3)(-). Two Escherichia coli PCK-CO(2) crystal structures are presented here. The location of CO(2) is the same for both structures; however the orientation of CO(2) is significantly different, likely from the presence of a manganese ion in one of the structures. PCK and the other three known protein-CO(2) crystal structure complexes have been compared; all have CO(2) hydrogen bonding with a basic amino acid side chain (Arg65 or Lys213 in PCK), likely to polarize CO(2) to make the central carbon atom more electrophilic and thus more reactive. Kinetic studies found that the PCK mutant Arg65Gln increased the K(M) for substrates PEP and oxaloacetate but not for CO(2). The unchanged K(M) for CO(2) can be explained since the Arg65Gln mutant likely maintains a hydrogen bond to one of the oxygen atoms of carbon dioxide.     

DataSpaces: an interaction and coordination framework for?coupled simulation workflows

Emerging high-performance distributed computing environments are enabling new end-to-end formulations in science and engineering that involve multiple interacting processes and data-intensive application workflows. For example, current fusion simulation efforts are exploring coupled models and codes that simultaneously simulate separate application processes, such as the core and the edge turbulence. These components run on different high performance computing resources, need to interact at runtime with each other and with services for data monitoring, data analysis and visualization, and data archiving. As a result, they require efficient and scalable support for dynamic and flexible couplings and interactions, which remains a challenge. This paper presents DataSpaces a flexible interaction and coordination substrate that addresses this challenge. DataSpaces essentially implements a semantically specialized virtual shared space abstraction that can be associatively accessed by all components and services in the application workflow. It enables live data to be extracted from running simulation components, indexes this data online, and then allows it to be monitored, queried and accessed by other components and services via the space using semantically meaningful operators. The underlying data transport is asynchronous, low-overhead and largely memory-to-memory. The design, implementation, and experimental evaluation of DataSpaces using a coupled fusion simulation workflow is presented.     

VEGF and bone cell signalling: an essential vessel for communication?

Vascular endothelial growth factor (VEGF) is an endothelial cell survival factor and is required for effective coupling of angiogenesis and osteogenesis. Although central to bone homeostasis, repair and the pathobiology that affect these processes, the precise mechanisms coupling endothelial cell function within bone formation and remodelling remain unclarified. This review will (i) focus on the potential directionality of VEGF signalling in adult bone by identifying the predominant source of VEGF within the bone microenvironment, (ii) will summarize current VEGF receptor expression studies by bone cells and (iii) will provide evidence for a role for VEGF signalling during postnatal repair and osteoporosis. A means of understanding the directionality of VEGF signalling in adult bone would allow us to most effectively target angiogenic pathways in diseases characterized by changes in bone remodelling rates and enhance bone repair when compromised. Copyright © 2012 John Wiley & Sons, Ltd.     

Selenium and diabetes: an enigma?

In recent years diabetes has become one of the most common metabolic diseases in developed countries and it is closely related to supernutrition and obesity. Since untreated diabetes produces oxidative stress responsible for secondary complications of the disease, antioxidant supplements were considered as being favourable for the therapy of diabetes. However, the situation has changed recently, since large cross-sectional and interventional trials revealed a positive correlation between a high Se status and diabetes incidence in humans. Thus, currently available data on the role of Se in diabetes are inconsistent and an enigma appears to exist for the relation between selenium and diabetes. This review summarizes selected human and animal studies, pointing to beneficial and critical virtues of Se in diabetes. Moreover, the review discusses possible underlying mechanisms how Se may influence diabetes in both directions. From the current literature, the following information can be extracted: (1) In populations with a high Se status, with the single exception of pregnant women, Se supplements cannot be recommended for the prevention of diabetes; (2) Anti-diabetic effects of Se seem to be restricted to high and nearly toxic doses which cannot be used in humans; and (3) Future investigations should consider the stage of the disease.     

Slowness: an objective for spike-timing-dependent plasticity?

Our nervous system can efficiently recognize objects in spite of changes in contextual variables such as perspective or lighting conditions. Several lines of research have proposed that this ability for invariant recognition is learned by exploiting the fact that object identities typically vary more slowly in time than contextual variables or noise. Here, we study the question of how this "temporal stability" or "slowness" approach can be implemented within the limits of biologically realistic spike-based learning rules. We first show that slow feature analysis, an algorithm that is based on slowness, can be implemented in linear continuous model neurons by means of a modified Hebbian learning rule. This approach provides a link to the trace rule, which is another implementation of slowness learning. Then, we show analytically that for linear Poisson neurons, slowness learning can be implemented by spike-timing-dependent plasticity (STDP) with a specific learning window. By studying the learning dynamics of STDP, we show that for functional interpretations of STDP, it is not the learning window alone that is relevant but rather the convolution of the learning window with the postsynaptic potential. We then derive STDP learning windows that implement slow feature analysis and the "trace rule." The resulting learning windows are compatible with physiological data both in shape and timescale. Moreover, our analysis shows that the learning window can be split into two functionally different components that are sensitive to reversible and irreversible aspects of the input statistics, respectively. The theory indicates that irreversible input statistics are not in favor of stable weight distributions but may generate oscillatory weight dynamics. Our analysis offers a novel interpretation for the functional role of STDP in physiological neurons.     

Is rat skeletal muscle hexokinase an allosteric enzyme?

A recent report by M. Gregoriou, I. P. Trayer, and A. Cornish-Bowden (1986, Eur. J. Biochem. 161, 171-176) showed that the mechanism for rat skeletal muscle hexokinase contains two allosteric sites: one for ATP and one for glucose 6-phosphate. In this report, we show that the allosteric mechanism is at variance with a large amount of kinetic data for the skeletal muscle hexokinase reaction in the literature. In addition, the allosteric mechanism conflicts with isotope exchange at chemical equilibrium data reported by M. Gregoriou, I. P. Trayer, and A. Cornish-Bowden (1983, Eur. J. Biochem. 134, 283-288).