Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

BACKGROUND: We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. RESULTS: Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP) and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5-7.0 and a high Km for ThDP (5 mM), suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC 2.7.7.65). CONCLUSION: This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells

Neuroprotective effects of thiamine and precursors with higher bioavailability: focus on benfotiamine and dibenzoylthiamine

Thiamine (vitamin B1) is essential for brain. This is attributed to the coenzyme role of thiamine diphosphate (ThDP) in glucose and energy metabolism. However, thiamine triphosphorylated derivatives thiamine triphosphate and adenosine thiamine triphosphate — the latter discovered in our laboratory — have been described, but heir physiological roles remain unknown. We have recently become interested in thiamine precursors with higher bioavailability. Among these, the thioester benfotiamine (BFT) has been extensively studied and has beneficial effects both in rodent models of neurodegeneration and in human clinical studies. BFT has no known adverse effects and improves cognitive outcome in patients with mild Alzheimer’s disease (AD). The mechanisms of action of BFT remains unknown. Indeed, in cell culture and animal models, BFT has antioxidant and anti-inflammatory properties that seem to be mediated by a mechanism independent of the coenzyme function of ThDP. Recent in vitro studies show that another thiamine thioester, dibenzoylthiamine (DBT) is even more efficient that BFT, especially with respect to its anti-inflammatory potency and is effective at lower concentrations. Thiamine thioesters have pleiotropic properties linked to an increase in circulating thiamine concentrations and possibly in hitherto unidentified open thiazole ring derivatives. The identification of the active neuroprotective derivatives and the clarification of their mechanism of action open extremely promising perspectives in the field of neurodegenerative, neurodevelopmental and psychiatric conditions

Reduced Nucleotides, Thiols and O2 in Cellular Redox Balance: a Biochemist’s View

peer reviewedIn the present review, which is aimed at researchers, teachers and students in life sciences, we try to show how the physicochemical properties of the elements and molecules define the concept of redox balance. Living organism are open systems traversed by fluxes of energy and matter. Dur-ing catabolic oxidative metabolism, matter, mostly hydrogenated organic molecules are oxi-dized and ultimately released as CO2. Electrons are passed over to coupling molecules, such as NAD+ and FAD, whose reduced forms serve as electrons donors in anabolic reactions. Early photosynthetic activity led to the accumulation of O2 and the transformation of the reducing to an oxidizing atmosphere, favoring the development of oxidative metabolism in living organisms. We focus on the specific properties of O2 which provides the chemical energy for the combustion reactions occurring in living cells. We explain the concepts of redox potential and redox balance in complex systems such as living cells and we present the main redox couples involved in cellu-lar redox balance and discuss the chemical properties underlying their cellular roles and, in par-ticular, their antioxidant properties in the defense against reactive oxygen species (ROS). Finally, we try to provide an integrative view emphasizing the interplay between metabolism, oxidative stress and metabolic compartmentation in mammalian cells

Molecular mechanisms of the non-coenzyme action of thiamin in brain. Biochemical, structural and pathway analysis

Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDPdependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins

El tercero del juego

Desde un análisis crítico, la autora afirma que la divulgación masiva de los avances de la tecnociencia, y el territorio del saber legítimo aparentan abrir sus fronteras al ciudadano, sin embargo la distancia entre el conocimiento general y el conocimiento especializado constituye una brecha que obtura la realización armónica del proceso social. En la búsqueda de una justificación tentativa para la imbricación entre teoría y praxis sitúa, desde un punto de vista semiótico la noción de relación, a través de un tercer elemento vinculante, la creatividad o la imaginación creadora. Sobre el foco de la creatividad sustenta la posibilidad del sujeto de indagar por sí mismo acerca de su entorno y sus condiciones de existencia.From a critical analyses the author affirms that the massive spreading of the advances of the technoscience, the territory of the legal knowledge seems to open its frontiers to the citizen, however the distance between the general knowledge and the specialized knowledge constitutes a breach that stops the harmonic fulfillment of the social process. In the searching of a tentative justification for the imbrication between theory and praxes it paces, from a semiotic point of view, the notion of relation, through a third associated element, the creativity or the creative imagination. Over the focus of the creativity it is maintained the possibility of the person of questioning by itself about its contour and its conditions of existence.Desde uma análise crítica o autor afirma que a divulgação de massa dos avanços da tecnociência, o território do saber legítimo aparentan abrir suas fronteiras ao cidadão,todavia a distância entre o conhecimento geral e o conhecimento especializado constitui umabrecha que obtura a realização harmônica do processo social. Na busca de uma justificaçãotentativa para a imbricação entre a teoria e a práxis situa, desde um ponto de vista semiótico, anoção de relação, através de um terceiro elemento vinculador, a criatividade ou a imaginaçãocriadora.Sob o foco da criatividade sustenta a possibilidade do sujeito de indagar por ele própriocom respeito a seu entorno e suas condições de existência

La imagen vigilante: acerca de la fotografía policial como instrumento del poder

Incorporated into the surveillance device of the State Repressive Apparatus, the character of evidence or evidence of the photographs of the police archives is the product of an institutional construction that converts into knowing what is initially perceived as the imprint of an event. In this article we are dedicated, precisely, to examine the functionality of this genre of images within the Directorate of Intelligence of the Police of the Province of Buenos –DIPBA– in the period 1962-1973. We also propose the concept of “scopic memory” to account for the way of seeing / showing / knowing proper to the institution in relation to its photographic production.Incorporadas al dispositivo de vigilancia del Aparato Represivo de Estado, el carácter de prueba o evidencia de las fotografías de los archivos policiales es producto de una construcción institucional que convierte en saber aquello que inicialmente se percibe como huella de un acontecer. En este artículo nos dedicamos, precisamente, a examinar la funcionalidad de este género de imágenes al interior de la Dirección de Inteligencia de la Policía de la Provincia de Buenos –DIPBA– en el periodo 1962-1973. Proponemos además el concepto de “memoria escópica” para dar cuenta del modo de ver/mostrar/saber propio de la institución en relación con su producción fotográfica.Incorporado ao dispositivo de vigilância do Aparelho Repressivo do Estado, o caráter de evidência ou evidência das fotografias dos arquivos policiais é o produto de uma construção institucional que se converte em saber o que é inicialmente percebido como a impressão de um evento. Neste artigo estamos dedicados, precisamente, a examinar a funcionalidade deste gênero de imagens dentro da Diretoria de Inteligência da Polícia da Província de Buenos - DIPBA - no período 1962-1973. Também propomos o conceito de “memória escópica” para explicar o modo de ver / mostrar / conhecer adequado à instituição em relação à sua produção fotográfica

Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coli

BACKGROUND: Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. RESULTS: Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine) at 37 degrees C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. CONCLUSION: These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis

Imagen y percepción. La apuesta por un realismo sinestésico en el Nuevo Cine Argentino realizado por mujeres.

El Nuevo Cine Argentino realizado por mujeres. ¿Cómo pensar el realismo en el Nuevo Cine Argentino? ¿Y cómo pensarlo específicamente para las producciones de algunas de las cineastas claves de este período? Estos interrogantes se desprenden de un proyecto más amplio que tiene por objeto el cine realizado por mujeres en Argentina 1: una investigación que podría considerarse acotada si se enfocara solamente en el cine producido en Argentina en el siglo XX, desde sus inicios, pasando por la época de los estudios, hasta los años 1990; y que se revela mucho más extensa a partir del cambio de siglo. El año 2000 marca un ingreso de mujeres al campo de la realización cinematográfica que se multiplica año a año. A partir de esta fecha se puede comprobar la aparición de directoras que ya no son responsables de un solo film, sino que empiezan a tener una “obra” (con todas las complejidades que implica este concepto, incluyendo la problemática del “autor”) y, sobre todo, una apuesta programática por una forma de cine

Adenosine thiamine triphosphate accumulates in Escherichia coli cells in response to specific conditions of metabolic stress

<p>Abstract</p> <p>Background</p> <p><it>E. coli </it>cells are rich in thiamine, most of it in the form of the cofactor thiamine diphosphate (ThDP). Free ThDP is the precursor for two triphosphorylated derivatives, thiamine triphosphate (ThTP) and the newly discovered adenosine thiamine triphosphate (AThTP). While, ThTP accumulation requires oxidation of a carbon source, AThTP slowly accumulates in response to carbon starvation, reaching ~15% of total thiamine. Here, we address the question whether AThTP accumulation in <it>E. coli </it>is triggered by the absence of a carbon source in the medium, the resulting drop in energy charge or other forms of metabolic stress.</p> <p>Results</p> <p>In minimal M9 medium, <it>E. coli </it>cells produce AThTP not only when energy substrates are lacking but also when their metabolization is inhibited. Thus AThTP accumulates in the presence of glucose, when glycolysis is blocked by iodoacetate, or in the presence lactate, when respiration is blocked by cyanide or anoxia. In both cases, ATP synthesis is impaired, but AThTP accumulation does not appear to be a direct consequence of reduced ATP levels. Indeed, in the CV2 <it>E. coli </it>strain (containing a thermolabile adenylate kinase), the ATP content is very low at 37°C, even in the presence of metabolizable substrates (glucose or lactate) and under these conditions, the cells produce ThTP but not AThTP. Furthermore, we show that ThTP inhibits AThTP accumulation. Therefore, we conclude that a low energy charge is not sufficient to trigger AThTP accumulation and the latter can only accumulate under conditions where no ThTP is synthesized. We further show that AThTP production can also be induced by the uncoupler CCCP but, unexpectedly, this requires the presence of pyruvate or a substrate yielding pyruvate (such a D-glucose or L-lactate). Under the conditions described, AThTP production is not different when RelA or SpoT mutants are used.</p> <p>Conclusions</p> <p>In <it>E. coli</it>, AThTP accumulates in response to two different conditions of metabolic stress: lack of energy substrates (or inhibition of their metabolization) and uncoupled pyruvate oxidation. Both conditions prevent bacterial growth. There is no obvious link with the stringent response or catabolite repression.</p

Dysautonomia, A Heuristic Approach to a Revised Model for Etiology of Disease

Dysautonomia refers to a disease where the autonomic nervous system is dysfunctional. This may be a central control mechanism, as in genetically determined familial dysautonomia (Riley-Day Syndrome), or peripherally in the distribution of the sympathetic and parasympathetic systems. There are multiple reports of a number of different diseases associated with dysautonomia. The etiology of this association has never been explained. There are also multiple publications on dysautonomia associated with specific non-caloric nutritional deficiencies. Beriberi is the prototype of autonomic dysfunction. It is the best known nutritional deficiency disease caused by an imbalance between ingested calories and the vitamins required for their oxidation, particularly thiamin. Long thought to be abolished in modern medical thinking, there are occasional isolated reports of the full-blown disease in developed Western cultures