Adaptations of skeletal muscle pyruvate dehydrogenase kinase in response to food-restriction in mitochondrial subpopulations

University, 2006 Dr. Sandra J. Peters Pyruvate dehydrogenase (PDH) catalyses the decarboxylation of pyruvate, to form acetyl-CoA. PDH activity is down-regulated by intrinsic PDH kinases (predominantly PDK2 and PDK4 isoforms), but the understanding of the PDK isoform distribution and adaptation to nutritional stresses has been restricted to mixed mitochondrial populations, and not delineated between subsarcolemmal (SS) and intermyofibrillar (IMF) subpopulations. SS and IMF mitochondria exhibit distinct morphological and biochemical properties; however the functional differences are not well understood. This study investigated the effect of fed (FED) versus 48 h total foodrestriction (FR) on rat red gastrocnemius muscle PDK2 and 4 isoform content in SS and IMF mitochondria. PDK4 content was ~3-5 fold higher in SS mitochondria compared to IMF (p=0.001), and increased with FR -3-4- fold in both subpopulations (p<0.001). PDK2 was -2.5-4 fold higher in SS mitochondria compared to IMF (p=0.001), but PDK2 was unaltered with FR. Citrate synthase activity (|imol/min/mg mitochondrial protein) was not different between either subpopulation. As well there were no significant differences between mitochondrial subpopulations in PDH complex components in both fed and FR states. These results demonstrate that there is a markedly higher content of both PDK isofonns in SS compared to IMF mitochondria. Although PDK2 does not increase in either subpopulation in response to FR, PDK4 increases to a similar extent in both SS and IMF after 48 h food-restriction.

An investigation of ethylene inhibition of growth in etiolated Avena sativa coleoptiles /

Growth rates of etiolated Avena sativa coleoptiles in pH 7.0 buffered medium are stimulated in a synergistic manner by IAA and 320 ~l/l carbon dioxide. The suggestion that carbon dioxide stimulated growth involves dark fixation is supported by the ability of 1 mM malate to replace carbon dioxide, with neither factor able to stimulate growth in the presence of the other (Bown, Dymock and Aung, 1974). The regulation of Avena coleoptile growth by ethylene has been investigated in the light of this data and the well documented antagonism between carbon dioxide and ethylene in the regulation of developmental processes. The influence of various permutations of ethylene, IAA, carbon dioxide and malate on the rates of growth, l4c-bicarbonate incorporation, l4C-bicarbonate fixation, and malate decarboxylation have been investigated. In the presence of 320 ~l/l carbon dioxide, 10.8 ~l/l ethylene inhibited growth both in the absence and presence of 20 ~M IAA with inhibition times, of 8-10 and 12-13 minutes respectively. In contrast ethylene inhibition of growth was not significant in the absence of growth stimulation by CO2 or 1 mM malate, and the normal growth increases in response to CO2 and malate were blocked by the simultaneous application of ethylene. The rates of incorporation and dark fixation of l4C-bicerbonate were not measurably. influenced by ethylene, IAA or malate, either prior to or during the changes in growth ,ates induced by these agents. The data does not support the hypothesis that ethylene inhibition of growth results from an inhibition of dark fixation, but suggests that ethylene may inhibit a process which is subsequent to fixation.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

Rapid [Gamma]-amino acid synthesis and the inhibition of the growth and development of oblique banded leaf-roller larvae

The hypothesis that rapid y-aminobutyric acid (GABA) accumulation is a plant defense against phytophagous insects was investigated. Simulation of mechanical damage resulting from phytophagous insect activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2. 15 (±O. 11 SE) ~mol GABA per gram fresh weight. Increasing the GABA levels in a synthetic diet from 1.6 to 2.6 Jlffiol GABA per gram fresh weight reduced the growth rates, developmental rates, total biomass (50% reduction), and survival rates (30% reduction) of cultured Oblique banded leaf-roller (OBLR) (Choristonellra rosacealla Harris) larvae. In field experiments OBLR larvae were found predominantly on young terminal leaves which have a reduced capacity to produce GABA in response to mechanical damage. Glutamate decarboxylase (GAD) is a cytosolic enzyme which catalyses the decarboxylation of L-Glu to GABA. GAD is a calmodulin binding enzyme whose activity is stimulated dramatically by increased cytosolic H+ or Ca2 + ion concentrations. Phytophagous insect activity will disrupt the cellular compartmentation of H+ and Ca2 +, activate GAD and subsequent GABA accumulation. In animals GABA is a major inhibitory neurotransmitter. The possible mechanisms resulting in GABA inhibited growth and development of insects are discussed.

The production of 4-adminobutyrate in response to treatments reducing cytosolic pH and the regulation of intracellular pH

GABA (4-aminobutyrate) is synthesized through the decarboxylation of LGlu- (L-Glu-+ H+ ---> GABA + C02), and compared to many free amino acids is present in high concentrations in plant cells. GABA levels rise rapidly and dramatically in response to varied stress conditions including anaerobiosis. Recent papers suggest that GABA production and associated H+ consumption are parts of a metabolic pH-stat mechanism which ameliorates the intracellular pH decline associated with anaerobiosis or other treatments. To test this hypothesis GABA production and efflux have been measured in isolated Asparagus sprengeri cells in response to three treatments which potentially cause intracellular acidification. Acid loads were imposed using 60 min of (i) anaerobiosis, (ii) H+/LGlu- cotransport, and (iii) treatment with permeant weak acids (butyric, acetic and propionic). Both intra- and extracellular GABA concentrations increased more than 100% after anaerobiosis, almost 1000% after H+/L-Glu- cotransport (light or dark) and almost 5000/0 after addition of 5 mM butyric acid at pH 5.0. HPLC analysis of amino acids indicates that as GABA concentrations increased in response to butyric acid addition, glutamate concentrations decreased. Time-course studies demonstrated that added butyric acid stimulates GABA production by 2800/0 within 15 seconds. A fluorescent determination of cytosolic pH indicates that addition of butyric or other weak acids resulted in a rapid reduction in cytosolic pH of 0.6 pH units. The half time for the response to butyric acid addition is 2.1 seconds, indicating that the decline in cytosolic pH is rapid enough to account for the rapid stimulation of GABA production. The acid load in response to butyric acid addition was assayed by measurements of 14C-butyric acid uptake. Calculations indicate that GABA production accounted for 45% of the imposed acid load. The biological significance of GABA efflux is not yet understood. The results support the original hypothesis suggesting a role for GABA production in cellular pH regulation.

Synthèse formelle de l’hormaomycine et de la bélactosine A par utilisation d’une réaction de cyclopropanation intramoléculaire catalysée par un complexe de rhodium (II)

Ce mémoire présente trois approches différentes vers la synthèse du 3–(trans–2–nitrocyclopropyl)alanine, un intermédiaire synthétique de la hormaomycine. Cette molécule naturelle démontre d’intéressantes activités biologiques et pharmacologiques. Il est intéressant de souligner que ce dérivé donne facilement accès au 3–(trans–2–aminocyclopropyl)alanine, unité centrale de la bélactosine A. Ce composé naturel possédant lui aussi d’intéressantes propriétés biologiques, plusieurs études relationnelles structures-activités menant à des dérivés plus actifs de cette molécule ont été entreprises, démontrant l’intérêt toujours présent de synthétiser de façon efficace et optimale ces dérivés cyclopropaniques. Une méthodologie développée au sein de notre groupe de recherche et basée sur une réaction de cyclopropanation intramoléculaire diastéréosélective sera mise à profit afin d’élaborer une nouvelle voie de synthèse aussi élégante qu’efficace à la construction du 3–(trans–2–nitrocyclopropyl) alanine. En utilisant un carbène de rhodium généré soit par la dégradation d’un dérivé diazoïque, soit par la formation d’un réactif de type ylure d’iodonium, une réaction de cyclopropanation diastéréosélective permettra la formation de deux autres centres contigus et ce, sans même utiliser d’auxiliaire ou de catalyseur énantioenrichis. Ensuite, un réarrangement intramoléculaire précédant deux réactions synchronisées d’ouverture de cycle et de décarboxylation permettront l’obtention du composé d’intérêt avec un rendement global convenable et en relativement peu d’étapes. De cette manière, la synthèse formelle de la bélactosine A et de l’hormaomycine a été effectuée. Cette synthèse se démarque des autres par l’utilisation d’une seule transformation catalytique énantiosélective.

Pineal indoleamine metabolism in pyridoxine-deficient rats

Pvridoxine deficiency causes physiologically significant decrease in brain serotonin (5-HT) due to decreased decarboxylation of 5- hvdroxvtrvptophan (5-HTP). We have examined the effect of pyridoxine deficiency on indoleamine metabolism in the pineal gland, a tissue with high indoleamine turnover. Adult male Sprague-Dawley rats were fed either a pyridoxine-supplemented or pyridoxinedeficient diet for 8 weeks. Pyridoxine deficiency did not alter the pattern of circadian rhythm of pineal 5-HT. 5-hvdroxvindoleacetic acid (5-HIAA), V-acetvlserotonin (NAS). and melatonin. However the levels of these compounds were significantly lower in the pineal glands of pyridoxine-deficient animals. Pineal 5-HTP levels were consistently higher in the pyridoxine-deficient animals and a conspicuous increase was noticed at 22.00 h. Increase in pineal NAS and melatonin levels caused by isoproterenol (5 mg kg at 17.00 h) were significantly lower (P < 0.05) in the pyridoxine-deficient animals. Treatment of pyridoxine-deficient rats with pvridoxine restored the levels of pineal 5-HT, 5-HIAA. NAS. and melatonin to values seen in pyridoxine-supplemented control animals. These results suggest that 5-HT availability could be an important factor in the regulation of the synthesis of pineal NAS and melatonin.

2(3H)-Furanones and related substrates: Synthetic strategies and selectivity profile in their reactivity

The synthesis and reactions of simple derivatives of 2(3H)- and 3(2H)furanones have attracted considerable attention in recent years, primarily in connection with development of routes to antitumor agents that contain this ring as central structural unit. They also serve as useful synthetic building blocks for lactones and furans and are the precursors of a wide variety of biologically important heterocyclic systems. Although a number of syntheses of furanones were known they were in many cases limited to specific substitution pattems. The development of altemative strategies for the preparation of these heterocycles is therefore of considerable importance or continues to be a challenge.We propose to develop new and general approaches to the synthesis of furanone ring systems from simple and readily available starting materials since we were interested in examining their rich photochemistry. The photochemical reactivity of Beta,gama-unsaturated lactams and lactones is a subject of current interest. Some of the prominent photoreaction pathways of unsaturated lactones include decarbonylation, solvent addition to double bonds, decarboxylation, migration of aryl substituents and dimerisation. lt was reported earlier that the critical requirement for clean photochemical cleavage of the acyl-oxygen bond is the presence ofa double bond adjacent to the ether oxygen and 2(3H)-furanones possessing this structural requirement undergo facile decarbonylation. But related phenanthrofuranones are isolated as photostable end products upon irradiation. Hence we propose to synthesis a few phenanthro-2(3H)-furanones to study the effect of a radical stabilising group at 3-position of furanone ring on photolysis. To explore the tripletmediated transformations of 2(3H)-furanones in polar and nonpolar solvents a few 3,3-bis(4-chlorophenyl)-5-aryl-3H-furan-2-ones and 3,3-di(p-tolyl)-5-aryl- 3H-furan-2-ones were synthesised from the corresponding dibenzoylstyrene precursors by neat thermolysis. Our aim was to study the nature of intermediates involved in these transformations.We also explored the possibility of developing a new and general approach to the synthesis of 3(2H)-furanones from simple and readily available starting materials since such general procedures are not available. The protocol developed by us employs readily available phenanthrenequinone and various 4-substituted acetophenones as starting materials and provides easy access to the required 3(2H)-furanone targets. These furanone derivatives have immense potential for further investigations .We also aimed the synthesis of a few dibenzoylalkene-type systems such as acenaphthenone-2—ylidene ketones and phenanthrenone-9-ylidene ketones. These systems were expected to undergo thermal rearrangement to give furanones and spirofuranones. Also these systems can be categorised as quinonemethides which are valuable synthetic intermediates.

Interaction of 2-(arylazo)phenols with rhodium. Usual coordination vs. C-H and C-C activation

Reaction of 2-(2'-hydroxyphenylazo)phenol with [Rh(PPh3)(3)Cl] in refluxing benzene in presence of triethylamine afforded a red complex in which the ligand is coordinated to rhodium as a tridentate O,N,O-donor. However, similar reaction of [Rh(PPh3)(3)Cl] with 2-(2'carboxyphenylazo)-4-methylphenol yielded two complexes, viz. a blue one and a green one. In both the complexes the ligand is coordinated as C,N,O-donor. However, in the blue complex orthometallation takes place from the ortho-carbon atom, which bears -COOH group via decarboxylation and in green one orthometallation occurs from the other ortho-carbon. Structures of all the three complexes were determined by X-ray crystallography. In all the three complexes rhodium is sharing the equatorial plane with the tridentate ligand and a chloride, and the two triphenylphosphines are axially disposed. All of the complexes show intense MLCT transitions in the visible region. Cyclic voltammetry on these complexes shows a Rh(III)-Rh(IV) oxidation on the positive side of SCE and a reduction of the coordinated azophenolate ligand on the negative side. (c) 2006 Elsevier B.V. All rights reserved.

Role of glutamate metabolism in bacterial responses towards acid and other stresses

Glutamate plays a central role in a wide range of metabolic processes in bacterial cells. This review focuses on the involvement of glutamate in bacterial stress responses. In particular it reviews the role of glutamate metabolism in response against acid stress and other stresses. The glutamate decarboxylase (GAD) system has been implicated in acid tolerance in several bacterial genera. This system facilitates intracellular pH homeostasis by consuming protons in a decarboxylation reaction that produces γ-aminobutyrate (GABA) from glutamate. An antiporter system is usually present to couple the uptake of glutamate to the efflux of GABA. Recent insights into the functioning of this system will be discussed. Finally the intracellular fate of GABA will also be discussed. Many bacteria are capable of metabolising GABA to succinate via the GABA shunt pathway. The role and regulation of this pathway will be addressed in the review. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S : roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.

An efficient and rapid microwave-assisted synthesis of 1-acetyl-1H-indol-3-YL acetates

An efficient and rapid synthesis of 1-acetyl-1H-indol-3-yl acetate 1 and its derivatives 7 via the microwave-assisted cyclisation and decarboxylation of 2-[(carboxymethyl)amino]benzoic acids 5 is described. The latter were left to react with acetic anhydride using triethylamine as the base and were subjected to microwave irradiation for 1 minute, at 80 °C with initial power of 300 W. The target 1-acetyl-1H-indol-3-yl acetate 1 and derivatives 7 were isolated in 34-71% yield. In particular, synthesis of 1-acetyl-6-(trifluoromethyl)-1H-indol-3-yl acetate 7f and 1-acetyl-3-methyl-1H-indol-3-yl acetate 7h is reported for the first time.

Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana

Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO(2) concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana: a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate. (C) 2010 Elsevier GmbH. All rights reserved.

Effect of exercise on glutamine metabolism in macrophages of trained rats

This study investigated the effect of exercise on glutamine metabolism in macrophages of trained rats. Rats were divided into three groups: sedentary (SED); moderately trained (MOD) rats that were swim trained 1 h/day, 5 days/week for 6 weeks; and exhaustively trained (EXT) rats that were similarly trained as MOD for 5 weeks and, in the 6th week, trained in three 1-h sessions/day with 150 min of rest between sessions. The animals swam with a load equivalent to 5.5% of their body weight and were killed 1 h after the last exercise session. Cells were collected, and glutamine metabolism in macrophage and function were assayed. Exercise increased phagocytosis in MOD when compared to SED (34.48 +/- 1.79 vs 15.21 +/- 2.91%, P < 0.05); however, H(2)O(2) production was higher in MOD (75.40 +/- 3.48 nmol h x 10(5) cell(-1)) and EXT (79.20 +/- 1.18 nmol h x 10(5) cell(-1)) in relation to SED (32.60 +/- 2.51 nmol h x 10(5) cell(-1), P < 0.05). Glutamine consumption increased in MOD and EXT (26.53 +/- 3.62 and 19.82 +/- 2.62 nmol h x 10(5) cell(-1), respectively) relative to SED (6.72 +/- 0.57 nmol h x 10(5) cell(-1), P < 0.05). Aspartate increased in EXT (9.72 +/- 1.14 nmol h x 10(5) cell(-1)) as compared to SED (1.10 +/- 0.19 nmol h x 10(5) cell(-1), P < 0.05). Glutamine decarboxylation was increased in MOD (12.10 +/- 0.27 nmol h x 10(5) cell(-1)) and EXT (16.40 +/-\ 2.17 nmol h x 10(5) cell(-1)) relative to SED (1.10 +/- 0.06 nmol h x 10(5) cell(-1), P < 0.05). This study suggests an increase in macrophage function post-exercise, which was supported by enhanced glutamine consumption and metabolism, and highlights the importance for glutamine after exercise.

Palythine-threonine, a major novel mycosporine-like amino acid (MAA) isolated from the hermatypic coral Pocillopora capitata

Using a high-resolution reverse-phase liquid chromatography method we found that the tissues of the hermatypic coral Pocillopora capitato (collected in Santiago Bay, Mexico) contain a high diversity of primary and secondary mycosporine-like amino acids (MAAs) typical of some reef-building coral species: mycosporine-glycine, shinorine, porphyra-334, mycosporine-methylamine-serine, mycosporine-methylamine-threonine, palythine-serine, palythine and one additional novel predominant MAA, with an absorbance maximum of 320 nm. Here we document the isolation and characterization of this novel MAA from the coral A capitata. Using low multi-stage mass analyses of deuterated and non deuterated compounds, high-resolution mass analyses (Time of Flight, TOF) and other techniques, this novel compound was characterized as palythine-threonine. Palythine-threonine was also present in high concentrations in the corals Pocillopora eydouxi and Stylophora pistillata indicating a wider distribution of this MAA among reef-building corals. From structural considerations we suggest that palythine-threonine is formed by decarboxylation of porphyra-334 followed by demethylation of mycosporine-methylamine-threonine. (C) 2008 Elsevier B.V. All rights reserved.