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Find video protocols related to scientific articles indexed in Pubmed.
A Consensus-based Gold Standard for the Evaluation of Mass Casualty Triage Systems.
Prehosp Emerg Care
PUBLISHED: 10-08-2014
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Abstract Introduction. Accuracy and effectiveness analyses of mass casualty triage systems are limited because there are no gold standard definitions for each of the triage categories. Until there is agreement on which patients should be identified by each triage category, it will be impossible to calculate sensitivity and specificity or to compare accuracy between triage systems. Objective. To develop a consensus-based, functional gold standard definition for each mass casualty triage category. Methods. National experts were recruited through the lead investigators' contacts and their suggested contacts. Key informant interviews were conducted to develop a list of potential criteria for defining each triage category. Panelists were interviewed in order of their availability until redundancy of themes was achieved. Panelists were blinded to each other's responses during the interviews. A modified Delphi survey was developed with the potential criteria identified during the interview and delivered to all recruited experts. In the early rounds, panelists could add, remove, or modify criteria. In the final rounds edits were made to the criteria until at least 80% agreement was achieved. Results. Thirteen national and local experts were recruited to participate in the project. Six interviews were conducted. Three rounds of voting were performed, with 12 panelists participating in the first round, 12 in the second round, and 13 in the third round. After the first two rounds, the criteria were modified according to respondent suggestions. In the final round, over 90% agreement was achieved for all but one criterion. A single e-mail vote was conducted on edits to the final criterion and consensus was achieved. Conclusion. A consensus-based, functional gold standard definition for each mass casualty triage category was developed. These gold standard definitions can be used to evaluate the accuracy of mass casualty triage systems after an actual incident, during training, or for research.
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Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate ?-Lyase Activity and Can Transaminate l-Selenomethionine.
J. Biol. Chem.
PUBLISHED: 09-17-2014
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Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), ?-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate ?-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no ?-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate ?-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate ?-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to ?-methylselenopyruvate (MSP) and ?-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-?-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the ?-elimination product of SM and the ?-elimination product of MSC catalyzed by cystathionine ?-lyase (?-cystathionase). KAT I, II, and III, in part, can catalyze ?-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine ?-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites.
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Thiosulfoxide (sulfane) sulfur: new chemistry and new regulatory roles in biology.
Molecules
PUBLISHED: 08-21-2014
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The understanding of sulfur bonding is undergoing change. Old theories on hypervalency of sulfur and the nature of the chalcogen-chalcogen bond are now questioned. At the same time, there is a rapidly expanding literature on the effects of sulfur in regulating biological systems. The two fields are inter-related because the new understanding of the thiosulfoxide bond helps to explain the newfound roles of sulfur in biology. This review examines the nature of thiosulfoxide (sulfane, S0) sulfur, the history of its regulatory role, its generation in biological systems, and its functions in cells. The functions include synthesis of cofactors (molybdenum cofactor, iron-sulfur clusters), sulfuration of tRNA, modulation of enzyme activities, and regulating the redox environment by several mechanisms (including the enhancement of the reductive capacity of glutathione). A brief review of the analogous form of selenium suggests that the toxicity of selenium may be due to over-reduction caused by the powerful reductive activity of glutathione perselenide.
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Arabidopsis and maize RidA proteins preempt reactive enamine/imine damage to branched-chain amino acid biosynthesis in plastids.
Plant Cell
PUBLISHED: 07-28-2014
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RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.
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Isolated loss of consciousness in children with minor blunt head trauma.
JAMA Pediatr
PUBLISHED: 07-09-2014
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A history of loss of consciousness (LOC) is frequently a driving factor for computed tomography use in the emergency department evaluation of children with blunt head trauma. Computed tomography carries a nonnegligible risk for lethal radiation-induced malignancy. The Pediatric Emergency Care Applied Research Network (PECARN) derived 2 age-specific prediction rules with 6 variables for clinically important traumatic brain injury (ciTBI), which included LOC as one of the risk factors.
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A comparison of reversible versus irreversible protein glutathionylation.
Adv. Cancer Res.
PUBLISHED: 06-30-2014
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Glutathionylation is generally a reversible posttranslational modification that occurs to cysteine residues that have been exposed to reactive oxygen species (P-SSG). This cyclical process can regulate various clusters of proteins, including those involved in critical cellular signaling functions. However, certain conditions can favor the formation of dehydroamino acids, such as 2,3-didehydroalanine (2,3-dehydroalanine, DHA) and 2,3-didehydrobutyrine (2,3-dehydrobutyrine), which can act as Michael acceptors. In turn, these can form Michael adducts with glutathione (GSH), resulting in the formation of a stable thioether conjugate, an irreversible process referred to as nonreducible glutathionylation. This is predicted to be prevalent in nature, particularly in more slowly turning over proteins. Such nonreducible glutathionylation can be distinguished from the more facile cycling signaling processes and is predicted to be of gerontological, toxicological, pharmacological, and oncological relevance. Here, we compare reversible and irreversible glutathionylation.
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The neurotoxicity of 5-S-cysteinyldopamine is mediated by the early activation of ERK1/2 followed by the subsequent activation of ASK1/JNK1/2 pro-apoptotic signalling.
Biochem. J.
PUBLISHED: 06-19-2014
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Parkinson's disease is characterized by the progressive and selective loss of dopaminergic neurons in the substantia nigra. It has been postulated that endogenously formed CysDA (5-S-cysteinyldopamine) and its metabolites may be, in part, responsible for this selective neuronal loss, although the mechanisms by which they contribute to such neurotoxicity are not understood. Exposure of neurons in culture to CysDA caused cell injury, apparent 12-48 h post-exposure. A portion of the neuronal death induced by CysDA was preceded by a rapid uptake and intracellular oxidation of CysDA, leading to an acute and transient activation of ERK2 (extracellular-signal-regulated kinase 2) and caspase 8. The oxidation of CysDA also induced the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser967, the phosphorylation of JNK (c-Jun N-terminal kinase) and c-Jun (Ser73) as well as the activation of p38, caspase 3, caspase 8, caspase 7 and caspase 9. Concurrently, the inhibition of complex I by the dihydrobenzothiazine DHBT-1 [7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid], formed from the intracellular oxidation of CysDA, induces complex I inhibition and the subsequent release of cytochrome c which further potentiates pro-apoptotic mechanisms. Our data suggest a novel comprehensive mechanism for CysDA that may hold relevance for the selective neuronal loss observed in Parkinson's disease.
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Occult pneumothoraces in children with blunt torso trauma.
Acad Emerg Med
PUBLISHED: 04-16-2014
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Plain chest x-ray (CXR) is often the initial screening test to identify pneumothoraces in trauma patients. Computed tomography (CT) scans can identify pneumothoraces not seen on CXR ("occult pneumothoraces"), but the clinical importance of these radiographically occult pneumothoraces in children is not well understood. The objectives of this study were to determine the proportion of occult pneumothoraces in injured children and the rate of treatment with tube thoracostomy among these children.
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A consensus-based criterion standard for trauma center need.
J Trauma Acute Care Surg
PUBLISHED: 03-26-2014
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In civilian trauma care, field triage is the process applied by prehospital care providers to identify patients who are likely to have severe injuries and immediately need the resources of a trauma center. Studies of the efficacy of field triage have used various measures to define trauma center need because no "criterion standard" exists, making cross-study comparisons difficult. This study aimed to develop a consensus-based functional criterion standard definition of trauma center need.
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From cholesterogenesis to steroidogenesis: role of riboflavin and flavoenzymes in the biosynthesis of vitamin D.
Adv Nutr
PUBLISHED: 03-13-2014
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Flavin-dependent monooxygenases and oxidoreductases are located at critical branch points in the biosynthesis and metabolism of cholesterol and vitamin D. These flavoproteins function as obligatory intermediates that accept 2 electrons from NAD(P)H with subsequent 1-electron transfers to a variety of cytochrome P450 (CYP) heme proteins within the mitochondria matrix (type I) and the (microsomal) endoplasmic reticulum (type II). The mode of electron transfer in these systems differs slightly in the number and form of the flavin prosthetic moiety. In the type I mitochondrial system, FAD-adrenodoxin reductase interfaces with adrenodoxin before electron transfer to CYP heme proteins. In the microsomal type II system, a diflavin (FAD/FMN)-dependent cytochrome P450 oxidoreductase [NAD(P)H-cytochrome P450 reductase (CPR)] donates electrons to a multitude of heme oxygenases. Both flavoenzyme complexes exhibit a commonality of function with all CYP enzymes and are crucial for maintaining a balance of cholesterol and vitamin D metabolites. Deficits in riboflavin availability, imbalances in the intracellular ratio of FAD to FMN, and mutations that affect flavin binding domains and/or interactions with client proteins result in marked structural alterations within the skeletal and central nervous systems similar to those of disorders (inborn errors) in the biosynthetic pathways that lead to cholesterol, steroid hormones, and vitamin D and their metabolites. Studies of riboflavin deficiency during embryonic development demonstrate congenital malformations similar to those associated with genetic alterations of the flavoenzymes in these pathways. Overall, a deeper understanding of the role of riboflavin in these pathways may prove essential to targeted therapeutic designs aimed at cholesterol and vitamin D metabolism.
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An approach for aggregating upstream catchment information to support research and management of fluvial systems across large landscapes.
Springerplus
PUBLISHED: 01-01-2014
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The growing quality and availability of spatial map layers (e.g., climate, geology, and land use) allow stream studies, which historically have occurred over small areas like a single watershed or stream reach, to increasingly explore questions from a landscape perspective. This large-scale perspective for fluvial studies depends on the ability to characterize influences on streams resulting from throughout entire upstream networks or catchments. While acquiring upstream information for a single reach is relatively straight-forward, this process becomes demanding when attempting to obtain summaries for all streams throughout a stream network and across large basins. Additionally, the complex nature of stream networks, including braided streams, adds to the challenge of accurately generating upstream summaries. This paper outlines an approach to solve these challenges by building a database and applying an algorithm to gather upstream landscape information for digitized stream networks. This approach avoids the need to directly use spatial data files in computation, and efficiently and accurately acquires various types of upstream summaries of landscape information across large regions using tabular processing. In particular, this approach is not limited to the use of any specific database software or programming language, and its flexibility allows it to be adapted to any digitized stream network as long as it meets a few minimum requirements. This efficient approach facilitates the growing demand of acquiring upstream summaries at large geographic scales and helps to support the use of landscape information in assisting management and decision-making across large regions.
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Terror medicine as part of the medical school curriculum.
Front Public Health
PUBLISHED: 01-01-2014
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Terror medicine, a field related to emergency and disaster medicine, focuses on medical issues ranging from preparedness to psychological manifestations specifically associated with terrorist attacks. Calls to teach aspects of the subject in American medical schools surged after the 2001 jetliner and anthrax attacks. Although the threat of terrorism persists, terror medicine is still addressed erratically if at all in most medical schools. This paper suggests a template for incorporating the subject throughout a 4-year medical curriculum. The instructional framework culminates in a short course for fourth year students, such as one recently introduced at Rutgers New Jersey Medical School, Newark, NJ, USA. The proposed 4-year Rutgers curriculum serves as a model that could assist other medical schools contemplating the inclusion of terror medicine in pre-clerkship and clerkship training.
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Lysine metabolism in mammalian brain: an update on the importance of recent discoveries.
Amino Acids
PUBLISHED: 08-28-2013
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The lysine catabolism pathway differs in adult mammalian brain from that in extracerebral tissues. The saccharopine pathway is the predominant lysine degradative pathway in extracerebral tissues, whereas the pipecolate pathway predominates in adult brain. The two pathways converge at the level of ?(1)-piperideine-6-carboxylate (P6C), which is in equilibrium with its open-chain aldehyde form, namely, ?-aminoadipate ?-semialdehyde (AAS). A unique feature of the pipecolate pathway is the formation of the cyclic ketimine intermediate ?(1)-piperideine-2-carboxylate (P2C) and its reduced metabolite L-pipecolate. A cerebral ketimine reductase (KR) has recently been identified that catalyzes the reduction of P2C to L-pipecolate. The discovery that this KR, which is capable of reducing not only P2C but also other cyclic imines, is identical to a previously well-described thyroid hormone-binding protein [?-crystallin (CRYM)], may hold the key to understanding the biological relevance of the pipecolate pathway and its importance in the brain. The finding that the KR activity of CRYM is strongly inhibited by the thyroid hormone 3,5,3-triiodothyronine (T3) has far-reaching biomedical and clinical implications. The inter-relationship between tryptophan and lysine catabolic pathways is discussed in the context of shared degradative enzymes and also potential regulation by thyroid hormones. This review traces the discoveries of enzymes involved in lysine metabolism in mammalian brain. However, there still remain unanswered questions as regards the importance of the pipecolate pathway in normal or diseased brain, including the nature of the first step in the pathway and the relationship of the pipecolate pathway to the tryptophan degradation pathway.
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?-Ketoglutaramate: an overlooked metabolite of glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle.
Metab Brain Dis
PUBLISHED: 08-14-2013
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Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to ?-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to ?-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ?-amidase. Transamination of glutamine results in formation of the corresponding ?-keto acid, namely, ?-ketoglutaramate (KGM). KGM is hydrolyzed by ?-amidase to ?-ketoglutarate and ammonia. The net glutaminase II reaction is: L?-?Glutamine?+???-?keto acid?+?H2O ? ??-?ketoglutarate?+?L?-?amino acid?+?ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance.
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Inhibition of human glutamine synthetase by L-methionine-S,R-sulfoximine-relevance to the treatment of neurological diseases.
Metab Brain Dis
PUBLISHED: 07-29-2013
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At high concentrations, the glutamine synthetase inhibitor L-methionine-S,R-sulfoximine (MSO) is a convulsant, especially in dogs. Nevertheless, sub-convulsive doses of MSO are neuroprotective in rodent models of hyperammonemia, acute liver disease, and amyotrophic lateral sclerosis and suggest MSO may be clinically useful. Previous work has also shown that much lower doses of MSO are required to produce convulsions in dogs than in primates. Evidence from the mid-20th century suggests that humans are also less sensitive. In the present work, the inhibition of recombinant human glutamine synthetase by MSO is shown to be biphasic-an initial reversible competitive inhibition (K i 1.19 mM) is followed by rapid irreversible inactivation. This K i value for the human enzyme accounts, in part, for relative insensitivity of primates to MSO and suggests that this inhibitor could be used to safely inhibit glutamine synthetase activity in humans.
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Structures of enzyme-intermediate complexes of yeast Nit2: insights into its catalytic mechanism and different substrate specificity compared with mammalian Nit2.
Acta Crystallogr. D Biol. Crystallogr.
PUBLISHED: 02-27-2013
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The Nit (nitrilase-like) protein subfamily constitutes branch 10 of the nitrilase superfamily. Nit proteins are widely distributed in nature. Mammals possess two members of the Nit subfamily, namely Nit1 and Nit2. Based on sequence similarity, yeast Nit2 (yNit2) is a homologue of mouse Nit1, a tumour-suppressor protein whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2 (also a putative tumour suppressor) is identical to ?-amidase, an enzyme that catalyzes the hydrolysis of ?-ketoglutaramate (?-KGM) and ?-ketosuccinamate (?-KSM) to ?-ketoglutarate (?-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with ?-KG and with OA were determined. In addition, the crystal structure of the C169S mutant of yNit2 (yNit2-C169S) in complex with an endogenous molecule of unknown structure was also solved. Analysis of the structures revealed that ?-KG and OA are covalently bound to Cys169 by the formation of a thioester bond between the sulfhydryl group of the cysteine residue and the ?-carboxyl group of ?-KG or the ?-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that ?-KGM is a relatively poor substrate of yNit2. Finally, a ligand was found in the active site of yNit2-C169S that may be a natural substrate of yNit2 or an endogenous regulator of enzyme activity. These crystallographic analyses provide information on the mode of substrate/ligand binding at the active site of yNit2 and insights into the catalytic mechanism. These findings suggest that yNit2 may have broad biological roles in yeast, especially in regard to nitrogen homeostasis, and provide a framework for the elucidation of the substrate specificity and biological role of mammalian Nit1.
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Imine Reductases: A Comparison of Glutamate Dehydrogenase to Ketimine Reductases in the Brain.
Neurochem. Res.
PUBLISHED: 01-12-2013
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A key intermediate in the glutamate dehydrogenase (GDH)-catalyzed reaction is an imine. Mechanistically, therefore, GDH exhibits similarities to the ketimine reductases. In the current review, we briefly discuss (a) the metabolic importance of the GDH reaction in liver and brain, (b) the mechanistic similarities between GDH and the ketimine reductases, (c) the metabolic importance of the brain ketimine reductases, and (d) the neurochemical consequences of defective ketimine reductases. Our review contains many historical references to the early work on amino acid metabolism. This work tends to be overlooked nowadays, but is crucial for a contemporary understanding of the central importance of ketimines in nitrogen and intermediary metabolism. The ketimine reductases are important enzymes linking nitrogen flow among several key amino acids, yet have been little studied. The cerebral importance of the ketimine reductases is an area of biomedical research that deserves far more attention.
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Quantitative Analysis of Neurotransmitter Pathways Under Steady State Conditions - A Perspective.
Front Endocrinol (Lausanne)
PUBLISHED: 01-01-2013
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In a contribution to this Research Topic Erkki Somersalo and Daniela Calvetti carried out a mathematical analysis of neurotransmitter pathways in brain, modeling compartmental nitrogen flux among several major participants - ammonia, glutamine, glutamate, GABA, and selected amino acids. This analysis is important because cerebral nitrogen metabolism is perturbed in many diseases, including liver disease and inborn errors of the urea cycle. These diseases result in an elevation of blood ammonia, which is neurotoxic. Here, a brief description is provided of the discovery of cerebral metabolic compartmentation of nitrogen metabolism - a key feature of cerebral glutamate-glutamine and GABA-glutamine cycles. The work of Somersalo and Calvetti is discussed as a model for future studies of normal and pathological cerebral ammonia metabolism.
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New insights into the metabolism of organomercury compounds: mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine ?-lyase.
Arch. Biochem. Biophys.
PUBLISHED: 08-22-2011
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Anthropogenic practices and recycling in the environment through natural processes result in release of potentially harmful levels of mercury into the biosphere. Mercury, especially organic forms, accumulates in the food chain. Mercury reacts readily with sulfur-containing compounds and often exists as a thiol S-conjugate, such as the l-cysteine (Cys)-S-conjugate of methylmercury (CH(3)Hg-S-Cys) or inorganic mercury (Cys-S-Hg-S-Cys). These S-conjugates are structurally similar to l-methionine and l-cystine/l-cystathionine, respectively. Bovine and rat glutamine transaminase K (GTK) catalyze transamination of sulfur-containing amino acids. Recombinant human GTK (rhGTK) has a relatively open catalytic active site, and we report here that this enzyme, like the rat and bovine enzymes, can also utilize sulfur-containing l-amino acids, including l-methionine, l-cystine, and l-cystathionine as substrates. The current study extends this list to include mercuric S-conjugates, and shows that CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are substrates and reversible inhibitors of rhGTK. The homocysteine S-conjugates, Hcy-S-Hg-S-Hcy and CH(3)Hg-S-Hcy, are also inhibitors. Finally, we show that HgCl(2), CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are potent irreversible inhibitors of rat cystathionine ?-lyase. The present study broadens our knowledge of the biochemistry of mercury compounds by showing that Cys S-conjugates of mercury interact with enzymes that catalyze transformations of biologically important sulfur-containing amino acids.
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The relationship between annual hospital volume of trauma patients and in-hospital mortality in New York State.
J Trauma
PUBLISHED: 08-10-2011
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Several studies in the literature have examined the volume-outcome relationship for trauma, but the findings have been mixed, and the associated impact of the trauma center level has not been examined to date. The purposes of this study are to (1) determine whether there is a significant relationship between the annual volume of trauma inpatients treated in a trauma center (with "patients" defined in multiple ways) and short-term mortality of those patients, and (2) examine the impact on the volume-mortality relationship of being a Level I versus Level II trauma center.
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Encephalopathy in acute liver failure resulting from acetaminophen intoxication: new observations with potential therapy.
Crit. Care Med.
PUBLISHED: 06-28-2011
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Hyperammonemia is a major contributing factor to the encephalopathy associated with liver disease. It is now generally accepted that hyperammonemia leads to toxic levels of glutamine in astrocytes. However, the mechanism by which excessive glutamine is toxic to astrocytes is controversial. Nevertheless, there is strong evidence that glutamine-induced osmotic swelling, especially in acute liver failure, is a contributing factor: the osmotic gliopathy theory. The object of the current communication is to present evidence for the osmotic gliopathy theory in a hyperammonemic patient who overdosed on acetaminophen.
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Reversible and irreversible protein glutathionylation: biological and clinical aspects.
Expert Opin Drug Metab Toxicol
PUBLISHED: 05-11-2011
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Depending in part on the glutathione:glutathione disulfide ratio, reversible protein glutathionylation to a mixed disulfide may occur. Reversible glutathionylation is important in protecting proteins against oxidative stress, guiding correct protein folding, regulating protein activity and modulating proteins critical to redox signaling. The potential also exists for irreversible protein glutathionylation via Michael addition of an -SH group to a dehydroalanyl residue, resulting in formation of a stable, non-reducible thioether linkage.
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The effect of observation on cranial computed tomography utilization for children after blunt head trauma.
Pediatrics
PUBLISHED: 05-09-2011
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Children with minor blunt head trauma often are observed in the emergency department before a decision is made regarding computed tomography use. We studied the impact of this clinical strategy on computed tomography use and outcomes.
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Mammalian forebrain ketimine reductase identified as ?-crystallin; potential regulation by thyroid hormones.
J. Neurochem.
PUBLISHED: 03-15-2011
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Ketimine reductase (E.C. 1.5.1.25) was purified to apparent homogeneity from lamb forebrain by means of a rapid multi-step chromatography protocol. The purified enzyme was identified by MS/MS (mass spectrometry) as ?-crystallin. The identity was confirmed by heterologously expressing human ?-crystallin in Escherichia coli and subsequent chromatographic purification of the protein. The purified human ?-crystallin was confirmed to have ketimine reductase activity with a maximum specific activity similar to that of native ovine ketimine reductase, and was found to catalyse a sequential reaction. The enzyme substrates are putative neuromodulator/transmitters. The thyroid hormone 3,5,3-l-triiodothyronine (T3) was found to be a strong reversible competitive inhibitor, and may have a novel role in regulating their concentrations. ?-Crystallin is also involved in intracellular T3 storage and transport. This research is the first to demonstrate an enzyme function for ?-crystallin. This newly demonstrated enzymatic activity identifies a new role for thyroid hormones in regulating mammalian amino acid metabolism, and a possible reciprocal role of enzyme activity regulating bioavailability of intracellular T3.
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A GC/MS-based metabolomic approach for diagnosing citrin deficiency.
Anal Bioanal Chem
PUBLISHED: 02-03-2011
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Citrin is the hepatic mitochondrial aspartate-glutamate carrier that is encoded by the gene SLC25A13. Citrin deficiency often leads to hyperammonemia, for which the current treatment concept is different from that for primary hyperammonemias. Metabolite level diagnosis, often referred to as chemical diagnosis, is not always successful in identifying citrin deficiency immediately or in a timely fashion. We previously made the chemical diagnosis of citrin deficiency in ten patients from nine families. In order to devise a more rapid and more accurate chemical diagnosis of this disorder than is currently available, we reinvestigated the gas chromatography/mass spectrometry-based urine metabolome in these patients. In patients aged 2 to 5 months, prominent biomarkers detected included one or more of the following metabolites: tyrosine, p-hydroxyphenyllactate, p-hydroxyphenylpyruvate, and N-acetyltyrosine, galactose, galactitol and galactonate, glucose, glucitol, and cystathionine. These biomarkers are less prominent in older patients, but are not increased in argininosuccinate synthetase deficiency or other hyperammonemias. ?-Ketoglutaramate (KGM), a recently recognized urinary biomarker of primary hyperammonemias associated with defects of the urea cycle, was increased in most patients with citrin deficiency studied here in spite of normal urinary levels of glutamine (the immediate precursor of KGM), 5-oxoproline, glutamate, aspartate, and asparagine. Other important urinary biomarkers that should be measured for differential diagnosis of hyperammonemias, including orotate, uracil, and ?-ureidopropionate, were not increased. The presence of citrulline and citrulline-derived metabolites was noted in all cases. The present study shows that noninvasive urine metabolomics, together with an analysis of selected metabolites or groups of metabolites, provides a more reliable and rapid chemical diagnosis of citrin deficiency than was previously available and more readily differentiates this disorder from other hyperammonemic syndromes.
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Urinary 2-hydroxy-5-oxoproline, the lactam form of ?-ketoglutaramate, is markedly increased in urea cycle disorders.
Anal Bioanal Chem
PUBLISHED: 01-16-2011
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?-Ketoglutaramate (KGM) is the ?-keto acid analogue of glutamine, which exists mostly in equilibrium with a lactam form (2-hydroxy-5-oxoproline) under physiological conditions. KGM was identified in human urine and its concentration quantified by gas chromatography/mass spectrometry (GC/MS). The keto acid was shown to be markedly elevated in urine obtained from patients with primary hyperammonemia due to an inherited metabolic defect in any one of the five enzymes of the urea cycle. Increased urinary KGM was also noted in other patients with primary hyperammonemia, including three patients with a defect resulting in lysinuric protein intolerance and one of two patients with a defect in the ornithine transporter I. These findings indicate disturbances in nitrogen metabolism, most probably at the level of glutamine metabolism in primary hyperammonemia diseases. Urinary KGM levels, however, were not well correlated with secondary hyperammonemia in patients with propionic acidemia or methylmalonic acidemia, possibly as a result, in part, of decreased glutamine levels. In conclusion, the GC/MS procedure has the required lower limit of quantification for analysis of urinary KGM, which is markedly increased in urea cycle disorders and other primary hyperammonemic diseases.
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Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 01-05-2011
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A synthetic polyanion composed of styrene, maleic anhydride, and methacrylic acid (molar ratio 56:37:7) significantly inhibited the respiration of isolated rat liver mitochondria in a time-dependent fashion that correlated with 1) collapse of the mitochondrial membrane potential and 2) high amplitude mitochondrial swelling. The process is apparently Ca(2+) dependent. Since it is blocked by cyclosporin A, the process is ascribed to induction of the mitochondrial permeability transition. In mitoplasts, i.e., mitochondria lacking their outer membranes, the polyanion rapidly blocked respiration. After incubation of rat liver mitochondria with the polyanion, cytochrome c was released into the incubation medium. In solution, the polyanion modified by conjugation with fluorescein formed a complex with cytochrome c. Addition of the polyanion to cytochrome c-loaded phosphatidylcholine/cardiolipin liposomes induced the release of the protein from liposomal membrane evidently due to coordinated interplay of Coulomb and hydrophobic interactions of the polymer with cytochrome c. We conclude that binding of the polyanion to cytochrome c renders it inactive in the respiratory chain due to exclusion from its native binding sites. Apparently, the polyanion interacts with cytochrome c in mitochondria and releases it to the medium through breakage of the outer membrane as a result of severe swelling. Similar properties were demonstrated for the natural polyanion, tobacco mosaic virus RNA. An electron microscopy study confirmed that both polyanions caused mitochondrial swelling. Exposure of cerebellar astroglial cells in culture to the synthetic polyanion resulted in cell death, which was associated with nuclear fragmentation.
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Measurement of cysteine S-conjugate ?-lyase activity.
Curr Protoc Toxicol
PUBLISHED: 10-16-2010
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Cysteine S-conjugate ?-lyases are pyridoxal 5-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH(2)CH(NH(3) (+))CO(2) (-)] and selenium Se-conjugates [RSeCH(2)CH(NH(3) (+))CO(2) (-)] that contain a leaving group in the ? position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. In mammals, at least ten PLP enzymes catalyze ?-elimination reactions with such cysteine S-conjugates. All are enzymes involved in amino acid metabolism that do not normally catalyze a ?-lyase reaction, but catalyze a non-physiological ?-lyase side-reaction that depends on the electron-withdrawing properties of the -SR or -SeR moiety. In the case of cysteine S-conjugates, if the eliminated RSH is stable, the compound may be S-thiomethylated and excreted (thiomethyl shunt) or S-glucuronidated and harmlessly excreted. However, if RSH is chemically reactive, the cysteine S-conjugate may be toxic as a result of the ?-lyase reaction. The cysteine S-conjugate ?-lyase pathway is of particular interest to toxicologists because it is involved in the bioactivation (toxification) of halogenated alkenes and certain drugs. This unit provides protocols for the analysis of cysteine S-conjugate ?-lyase activity.
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13N as a tracer for studying glutamate metabolism.
Neurochem. Int.
PUBLISHED: 09-13-2010
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This mini-review summarizes studies my associates and I carried out that are relevant to the topic of the present volume [i.e. glutamate dehydrogenase (GDH)] using radioactive (13)N (t(1/2) 9.96 min) as a biological tracer. These studies revealed the previously unrecognized rapidity with which nitrogen is exchanged among certain metabolites in vivo. For example, our work demonstrated that (a) the t(1/2) for conversion of portal vein ammonia to urea in the rat liver is ?10-11s, despite the need for five enzyme-catalyzed steps and two mitochondrial transport steps, (b) the residence time for ammonia in the blood of anesthetized rats is ?7-8s, (c) the t(1/2) for incorporation of blood-borne ammonia into glutamine in the normal rat brain is <3s, and (d) equilibration between glutamate and aspartate nitrogen in rat liver is extremely rapid (seconds), a reflection of the fact that the components of the hepatic aspartate aminotransferase reaction are in thermodynamic equilibrium. Our work emphasizes the importance of the GDH reaction in rat liver as a conduit for dissimilating or assimilating ammonia as needed. In contrast, our work shows that the GDH reaction in rat brain appears to operate mostly in the direction of ammonia production (dissimilation). The importance of the GDH reaction as an endogenous source of ammonia in the brain and the relation of GDH to the brain glutamine cycle is discussed. Finally, our work integrates with the increasing use of positron emission tomography (PET) and nuclear magnetic resonance (NMR) to study brain ammonia uptake and brain glutamine, respectively, in normal individuals and in patients with liver disease or other diseases associated with hyperammonemia.
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Inhibition of transglutaminase 2 mitigates transcriptional dysregulation in models of Huntington disease.
EMBO Mol Med
PUBLISHED: 07-29-2010
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Caused by a polyglutamine expansion in the huntingtin protein, Huntingtons disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de-repressed two established regulators of mitochondrial function, PGC-1alpha and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3-nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC-independent epigenetic strategy for treating neurodegeneration.
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Astrocyte glutamine synthetase: importance in hyperammonemic syndromes and potential target for therapy.
Neurotherapeutics
PUBLISHED: 01-21-2010
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Many theories have been advanced to explain the encephalopathy associated with chronic liver disease and with the less common acute form. A major factor contributing to hepatic encephalopathy is hyperammonemia resulting from portacaval shunting and/or liver damage. However, an increasing number of causes of hyperammonemic encephalopathy have been discovered that present with the same clinical and laboratory features found in acute liver failure, but without liver failure. Here, we critically review the physiology, pathology, and biochemistry of ammonia (i.e., NH3 plus NH4+) and show how these elements interact to constitute a syndrome that clinicians refer to as hyperammonemic encephalopathy (i.e., acute liver failure, fulminant hepatic failure, chronic liver disease). Included will be a brief history of the status of ammonia and the centrality of the astrocyte in brain nitrogen metabolism. Ammonia is normally detoxified in the liver and extrahepatic tissues by conversion to urea and glutamine, respectively. In the brain, glutamine synthesis is largely confined to astrocytes, and it is generally accepted that in hyperammonemia excess glutamine compromises astrocyte morphology and function. Mechanisms postulated to account for this toxicity will be examined with emphasis on the osmotic effects of excess glutamine (the osmotic gliopathy theory). Because hyperammonemia causes osmotic stress and encephalopathy in patients with normal or abnormal liver function alike, the term "hyperammonemic encephalopathy" can be broadly applied to encephalopathy resulting from liver disease and from various other diseases that produce hyperammonemia. Finally, the possibility that a brain glutamine synthetase inhibitor may be of therapeutic benefit, especially in the acute form of liver disease, is discussed.
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Chemopreventive mechanisms of ?-keto acid metabolites of naturally occurring organoselenium compounds.
Amino Acids
PUBLISHED: 01-05-2010
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Previous studies on the chemopreventive mechanisms of dietary selenium have focused on its incorporation into antioxidative selenoproteins, such as glutathione peroxidase and thioredoxin reductase. Several studies, however, have revealed that dietary selenium in the form of L-selenomethionine and the 21st amino acid, selenocysteine, also have intrinsic anti-cancer properties. Biochemical mechanisms previously investigated to contribute to their anticancer effects involve ?- and ?-lyase reactions. Some pyridoxal 5-phosphate (PLP)-containing enzymes can catalyze a ?-lyase reaction with Se-methyl-L-selenocysteine (MSC) generating pyruvate and ammonia. Other PLP-enzymes can catalyze a ?-lyase reaction with L-selenomethionine (SM) generating ?-ketobutyrate and ammonia. In both cases, a purported third product is methylselenol (CH(3)SeH). Although not directly quantifiable, as a result of its extreme hydrophobicity and high vapor pressure, CH(3)SeH has been indirectly observed to act through the alteration of protein-sulfhydryl moieties on redox-responsive signal and transcription factors, thereby maintaining a non-proliferative intracellular environment. We have considered the possibility that ?-keto acid analogues of MSC (i.e., methylselenopyruvate; MSP) and SM (i.e., ?-keto-?-methylselenobutyrate; KMSB), generated via a transamination and/or L-amino acid oxidase reaction may also be chemoprotective. Indeed, these compounds were shown to increase the level of histone-H3 acetylation in human prostate and colon cancer cells. MSP and KMSB structurally resemble butyrate, an inhibitor of several histone deacetylases. Thus, the seleno ?-keto acid metabolites of MSC and SM, along with CH(3)SeH derived from ?- and ?-lyase reactions, may be potential direct-acting metabolites of organoselenium that lead to de-repression of silenced tumor suppressor proteins and/or regulation of genes and signaling molecules.
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Cysteine S-conjugate ?-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents.
Amino Acids
PUBLISHED: 01-05-2010
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Cysteine S-conjugate ?-lyases are pyridoxal 5-phosphate-containing enzymes that catalyze ?-elimination reactions with cysteine S-conjugates that possess a good leaving group in the ?-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate ?-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze ?-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate ?-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate ?-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1-15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M(r) cysteine S-conjugate ?-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic ?-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate ?-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate ?-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate ?-lyases are biologically more important than hitherto appreciated.
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Minocycline chelates Ca2+, binds to membranes, and depolarizes mitochondria by formation of Ca2+-dependent ion channels.
J. Bioenerg. Biomembr.
PUBLISHED: 01-03-2010
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Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca(2+)-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca(2+) ions. Secondly, minocycline, in a Ca(2+)-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca(2+), induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca(2+)-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition.
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Mass critical care: pediatric considerations in extending and rationing care in public health emergencies.
Disaster Med Public Health Prep
PUBLISHED: 10-02-2009
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This article applies developing concepts of mass critical care (MCC) to children. In public health emergencies (PHEs), MCC would improve population outcomes by providing lifesaving interventions while delaying less urgent care. If needs exceed resources despite MCC, then rationing would allocate interventions to those most likely to survive with care. Gaps between estimated needs and actual hospital resources are worse for children than adults. Clear identification of pediatric hospitals would facilitate distribution of children according to PHE needs, but all hospitals must prepare to treat some children. Keeping children with a family member and identifying unaccompanied children complicate PHE regional triage. Pediatric critical care experts would teach and supervise supplemental providers. Adapting nearly equivalent equipment compensates for shortages, but there is no substitute for age-appropriate resuscitation masks, IV/suction catheters, endotracheal/gastric/chest tubes. Limitations will be encountered using adult ventilators for infants. Temporary manual bag valve ventilation and development of shared ventilators may prolong survival until the arrival of ventilator stockpiles. To ration MCC to children most likely to survive, the Pediatric Index of Mortality 2 score meets the criteria for validated pediatric mortality predictions. Policymakers must define population outcome goals in regard to lives saved versus life-years saved.
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Transglutaminase activation in neurodegenerative diseases.
Future Neurol
PUBLISHED: 08-14-2009
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The following review examines the role of calcium in promoting the in vitro and in vivo activation of transglutaminases in neurodegenerative disorders. Diseases such as Alzheimers disease, Parkinsons disease and Huntingtons disease exhibit increased transglutaminase activity and rises in intracellular calcium concentrations, which may be related. The aberrant activation of transglutaminase by calcium is thought to give rise to a variety of pathological moieties in these diseases, and the inhibition has been shown to have therapeutic benefit in animal and cellular models of neurodegeneration. Given the potential clinical relevance of transglutaminase inhibitors, we have also reviewed the recent development of such compounds.
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Alpha-keto acid metabolites of naturally occurring organoselenium compounds as inhibitors of histone deacetylase in human prostate cancer cells.
Cancer Prev Res (Phila)
PUBLISHED: 07-09-2009
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Histone deacetylase (HDAC) inhibitors are gaining interest as cancer therapeutic agents. We tested the hypothesis that natural organoselenium compounds might be metabolized to HDAC inhibitors in human prostate cancer cells. Se-Methyl-L-selenocysteine (MSC) and selenomethionine are amino acid components of selenium-enriched yeast. In a cell-free system, glutamine transaminase K (GTK) and L-amino acid oxidase convert MSC to the corresponding alpha-keto acid, beta-methylselenopyruvate (MSP), and L-amino acid oxidase converts selenomethionine to its corresponding alpha-keto acid, alpha-keto-gamma-methylselenobutyrate (KMSB). Although methionine (sulfur analogue of selenomethionine) is an excellent substrate for GTK, selenomethionine is poorly metabolized. Structurally, MSP and KMSB resemble the known HDAC inhibitor butyrate. We examined androgen-responsive LNCaP cells and androgen-independent LNCaP C4-2, PC-3, and DU145 cells and found that these human prostate cancer cells exhibit endogenous GTK activities. In the corresponding cytosolic extracts, the metabolism of MSC was accompanied by the concomitant formation of MSP. In MSP-treated and KMSB-treated prostate cancer cell lines, acetylated histone 3 levels increased within 5 hours, and returned to essentially baseline levels by 24 hours, suggesting a rapid, transient induction of histone acetylation. In an in vitro HDAC activity assay, the selenoamino acids, MSC and selenomethionine, had no effect at concentrations up to 2.5 mmol/L, whereas MSP and KMSB both inhibited HDAC activity. We conclude that, in addition to targeting redox-sensitive signaling proteins and transcription factors, alpha-keto acid metabolites of MSC and selenomethionine can alter HDAC activity and histone acetylation status. These findings provide a potential new paradigm by which naturally occurring organoselenium might prevent the progression of human prostate cancer.
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Identification of the putative tumor suppressor Nit2 as omega-amidase, an enzyme metabolically linked to glutamine and asparagine transamination.
Biochimie
PUBLISHED: 06-15-2009
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The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is omega-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated omega-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are alpha-ketoglutaramate and alpha-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of alpha-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating alpha-keto-gamma-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess alpha-ketoglutaramate may be neurotoxic. Moreover, alpha-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of omega-amidase is to remove potentially toxic intermediates by converting alpha-ketoglutaramate and alpha-ketosuccinamate to biologically useful alpha-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of omega-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology.
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Transglutaminases and neurodegeneration.
J. Neurochem.
PUBLISHED: 04-28-2009
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Transglutaminases (TGs) are Ca2+-dependent enzymes that catalyze a variety of modifications of glutaminyl (Q) residues. In the brain, these modifications include the covalent attachment of a number of amine-bearing compounds, including lysyl (K) residues and polyamines, which serve to either regulate enzyme activity or attach the TG substrates to biological matrices. Aberrant TG activity is thought to contribute to Alzheimer disease, Parkinson disease, Huntington disease, and supranuclear palsy. Strategies designed to interfere with TG activity have some benefit in animal models of Huntington and Parkinson diseases. The following review summarizes the involvement of TGs in neurodegenerative diseases and discusses the possible use of selective inhibitors as therapeutic agents in these diseases.
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Assay and purification of omega-amidase/Nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the alpha-keto acid analogues of glutamine and asparagine.
Anal. Biochem.
PUBLISHED: 04-13-2009
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omega-Amidase (omega-amidodicarboxylate amidohydrolase, EC 3.5.1.3) isolated from rat liver cytosol is a versatile enzyme that catalyzes a large number of amidase, transamidase, and ester hydrolysis reactions. omega-Amidase activity toward alpha-ketoglutaramate and alpha-ketosuccinamate (the alpha-keto acid analogues of glutamine and asparagine, respectively) is present in mammalian tissues, tumors, plants, bacteria, and fungi. Despite its versatility, widespread occurrence, and high specific activity, the enzyme has been little studied, possibly because the assay procedure previously required a substrate (alpha-ketoglutaramate) that is not commercially available. Here we report a simplified method for preparing alpha-ketoglutaramate and an assay procedure that measures alpha-ketoglutarate formation from alpha-ketoglutaramate in a 96-well plate format. We also describe a 96-well plate assay procedure that measures omega-amidase-catalyzed hydroxaminolysis of commercially available succinamic acid. The product, succinyl hydroxamate, yields a stable brown color in the presence of acidic ferric chloride that can be quantitated spectrophotometrically with negligible background interference. The two assay procedures (hydrolysis of alpha-ketoglutaramate and hydroxaminolysis of succinamate) were employed in purifying omega-amidase approximately 3600-fold from rat liver cytosol. The ratio of alpha-ketoglutaramate hydrolysis to succinamate hydroxaminolysis remained constant during the purification. omega-Amidase has recently been shown to be identical to Nit2, a putative tumor suppressor protein. It is anticipated that these new assay procedures will help to characterize the function of omega-amidase/Nit2 in tumor suppression, will provide the basis of high-throughput procedures to search for potent inhibitors and enhancers of omega-amidase, and will assist in identifying biological interactions between nitrogen metabolism and tumor biology.
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Developing consensus on appropriate standards of disaster care for children.
Disaster Med Public Health Prep
PUBLISHED: 03-19-2009
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Neither professional consensus nor evidence exists to guide the choice of essential hospital disaster interventions. The objective of our study was to demonstrate a method for developing consensus on hospital disaster interventions that should be regarded as essential, quantitatively balancing needs and resources.
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Measurement of sulfur-containing compounds involved in the metabolism and transport of cysteamine and cystamine. Regional differences in cerebral metabolism.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 02-27-2009
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An HPLC method with coulometric detection is presented for the quantitation of cysteamine, cystamine, thialysine, glutathione, glutathione disulfide and an oxidized metabolite of thialysine [S-(2-aminoethyl)-L-cysteine ketimine decarboxylated dimer (AECK-DD)]. The advantage of coulometric detection is that derivatization is unnecessary if the analyte is redox sensitive. The method was used to quantitate several sulfur-containing compounds in plasma and brain following gavage feeding of cysteamine to rats. Cysteamine, cystamine, thialysine and AECK-DD were detected in the brains of these animals. Interestingly, cysteamine treatment resulted in greatly elevated levels of cerebral methionine, despite the fact that cysteamine is not a precursor of methionine.
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A nonradioactive dot blot assay for transglutaminase activity.
Anal. Biochem.
PUBLISHED: 02-05-2009
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Aberrant transglutaminase (TG) activity has been implicated in the pathology of numerous diseases, including Huntingtons disease and Alzheimers disease. To fully characterize the role of TGs in these disorders, it is important that simple quantifiable assays be made available. The most commonly used assay currently employed requires significant time and a radioactive substrate. The assay described here uses a biotinylated substrate in conjunction with a dot blot apparatus to eliminate the use of radioactive substrates and allows relative transglutaminase activity to be measured simultaneously with minimal sample preparation in a large number of samples containing purified enzyme, cell extracts, or tissue homogenates.
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Guidelines for field triage of injured patients. Recommendations of the National Expert Panel on Field Triage.
MMWR Recomm Rep
PUBLISHED: 01-24-2009
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In the United States, injury is the leading cause of death for persons aged 1--44 years, and the approximately 800,000 emergency medical services (EMS) providers have a substantial impact on the care of injured persons and on public health. At an injury scene, EMS providers determine the severity of injury, initiate medical management, and identify the most appropriate facility to which to transport the patient through a process called "field triage." Although basic emergency services generally are consistent across hospital emergency departments (EDs), certain hospitals have additional expertise, resources, and equipment for treating severely injured patients. Such facilities, called "trauma centers," are classified from Level I (centers providing the highest level of trauma care) to Level IV (centers providing initial trauma care and transfer to a higher level of trauma care if necessary) depending on the scope of resources and services available. The risk for death of a severely injured person is 25% lower if the patient receives care at a Level I trauma center. However, not all patients require the services of a Level I trauma center; patients who are injured less severely might be served better by being transported to a closer ED capable of managing milder injuries. Transferring all injured patients to Level I trauma centers might overburden the centers, have a negative impact on patient outcomes, and decrease cost effectiveness. In 1986, the American College of Surgeons developed the Field Triage Decision Scheme (Decision Scheme), which serves as the basis for triage protocols for state and local EMS systems across the United States. The Decision Scheme is an algorithm that guides EMS providers through four decision steps (physiologic, anatomic, mechanism of injury, and special considerations) to determine the most appropriate destination facility within the local trauma care system. Since its initial publication in 1986, the Decision Scheme has been revised four times. In 2005, with support from the National Highway Traffic Safety Administration, CDC began facilitating revision of the Decision Scheme by hosting a series of meetings of the National Expert Panel on Field Triage, which includes injury-care providers, public health professionals, automotive industry representatives, and officials from federal agencies. The Panel reviewed relevant literature, presented its findings, and reached consensus on necessary revisions. The revised Decision Scheme was published in 2006. This report describes the process and rationale used by the Expert Panel to revise the Decision Scheme.
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Survey of trauma registry data on tourniquet use in pediatric war casualties.
Pediatr Emerg Care
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Previously, we reported on the use of emergency tourniquets to stop bleeding in war casualties, but virtually all the data were from adults. Because no pediatric-specific cohort of casualties receiving emergency tourniquets existed, we aimed to fill knowledge gaps on the care and outcomes of this group by surveying data from a trauma registry to refine device designs and clinical training.
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Enzymatic analysis of ?-ketoglutaramate--a biomarker for hyperammonemia.
Talanta
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Two enzymatic assays were developed for the analysis of ?-ketoglutaramate (KGM)-an important biomarker of hepatic encephalopathy and other hyperammonemic diseases. In both procedures, KGM is first converted to ?-ketoglutarate (KTG) via a reaction catalyzed by ?-amidase (AMD). In the first procedure, KTG generated in the AMD reaction initiates a biocatalytic cascade in which the concerted action of alanine transaminase and lactate dehydrogenase results in the oxidation of NADH. In the second procedure, KTG generated from KGM is reductively aminated, with the concomitant oxidation of NADH, in a reaction catalyzed by L-glutamic dehydrogenase. In both assays, the decrease in optical absorbance (?=340 nm) corresponding to NADH oxidation is used to quantify concentrations of KGM. The two analytical procedures were applied to 50% (v/v) human serum diluted with aqueous solutions containing the assay components and spiked with concentrations of KGM estimated to be present in normal human plasma and in plasma from hyperammonemic patients. Since KTG is the product of AMD-catalyzed hydrolysis of KGM, in a separate study, this compound was used as a surrogate for KGM. Statistical analyses of samples mimicking the concentration of KGM assumed to be present in normal and pathological concentration ranges were performed. Both enzymatic assays for KGM were confirmed to discriminate between the predicted normal and pathophysiological concentrations of the analyte. The present study is the first step toward the development of a clinically useful probe for KGM analysis in biological fluids.
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Possible treatment of end-stage hyperammonemic encephalopathy by inhibition of glutamine synthetase.
Metab Brain Dis
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Glutamine synthetase (GS) is highly active in astrocytes, and these cells are physiologically and morphologically compromised by hyperammonemia. Hyperammonemia in end-stage acute liver failure (ALF) is often associated with cerebral edema and astrocyte pathology/swelling. Many studies of animal models of hyperammonemia, and, more recently, nuclear magnetic resonance studies of liver disease patients, have shown that cerebral glutamine is elevated in hyperammonemia, contributing to the edema and encephalopathy. The GS inhibitor L-methionine-S,R-sulfoximine (MSO) is protective in animal models against acute ammonia intoxication. MSO is also an inhibitor of glutamate cysteine ligase, is converted to metabolic products, and causes convulsions at high doses. However, the susceptibility to MSO-induced convulsions is species dependent, with primates being relatively resistant. Moreover, it is possible to chronically maintain cerebral GS activity in mice at low levels by MSO treatment without any obvious untoward effects. Furthermore, MSO is protective in a mouse model of ALF. Extreme caution would be needed in administering MSO to patients. Nevertheless, inhibition of brain GS by MSO (or other GS inhibitors) may have therapeutic benefit in ALF.
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Burn disaster response planning in New York City: updated recommendations for best practices.
J Burn Care Res
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Since its inception in 2006, the New York City (NYC) Task Force for Patients with Burns has continued to develop a city-wide and regional response plan that addressed the triage, treatment, transportation of 50/million (400) adult and pediatric victims for 3 to 5 days after a large-scale burn disaster within NYC until such time that a burn center bed and transportation could be secured. The following presents updated recommendations on these planning efforts. Previously published literature, project deliverables, and meeting documents for the period of 2009-2010 were reviewed. A numerical simulation was designed to evaluate the triage algorithm developed for this plan. A new, secondary triage scoring algorithm, based on co-morbidities and predicted outcomes, was created to prioritize multiple patients within a given acuity and predicted survivability cohort. Recommendations for a centralized patient and resource tracking database, plan operations, activation thresholds, mass triage, communications, data flow, staffing, resource utilization, provider indemnification, and stakeholder roles and responsibilities were specified. Educational modules for prehospital providers and nonburn center nurses and physicians who would provide interim care to burn injured disaster victims were created and pilot tested. These updated best practice recommendations provide a strong foundation for further planning efforts, and as of February 2011, serve as the frame work for the NYC Burn Surge Response Plan that has been incorporated into the New York State Burn Plan.
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Cranial computed tomography use among children with minor blunt head trauma: association with race/ethnicity.
Arch Pediatr Adolesc Med
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To determine if patient race/ethnicity is independently associated with cranial computed tomography (CT) use among children with minor blunt head trauma.
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Stable isotope gas chromatography-tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples.
Anal. Biochem.
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Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD; systematic name: 1,2-3,4-5,6-7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one) is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, and cells in culture and vegetables and to possess potent antioxidative properties. Here, we describe a stable isotope gas chromatography-tandem mass spectrometry (GC-MS/MS) method for specific and sensitive determination of AECK-DD in biological samples. (13)C(2)-labeled AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected reaction monitoring of the mass transitions m/z 328 to 268 for AECK-DD and m/z 330 to 270 for [(13)C(2)]AECK-DD in the electron capture negative ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above approximately 4nM but was present in urine samples of healthy humans at a maximal concentration of 46nM. AECK-DD was detectable in rat brain at very low levels of approximately 8pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (?1nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (?6.8pmol/g fresh tissue).
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New mechanistic explanation for the localization of ulcers in the rat duodenum: role of iron and selective uptake of cysteamine.
Arch. Biochem. Biophys.
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Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.
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Structural insights into the catalytic active site and activity of human Nit2/?-amidase: kinetic assay and molecular dynamics simulation.
J. Biol. Chem.
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Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ?-amidase. hNit2/?-amidase plays a crucial metabolic role by catalyzing the hydrolysis of ?-ketoglutaramate (the ?-keto analog of glutamine) and ?-ketosuccinamate (the ?-keto analog of asparagine), yielding ?-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and ?-keto-?-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/?-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/?-amidase, we performed molecular dynamics simulations on the wild type enzyme and its mutants to investigate enzyme-substrate interactions. Binding free energies were computed to characterize factors contributing to the substrate specificity. The predictions resulting from these computations were verified by kinetic analyses and mutational studies. The activity of hNit2/?-amidase was determined with ?-ketoglutaramate and succinamate as substrates. We constructed three catalytic triad mutants (E43A, K112A, and C153A) and a mutant with a loop 116-128 deletion to validate the role of key residues and the 116-128 loop region in substrate binding and turnover. The molecular dynamics simulations successfully verified the experimental trends in the binding specificity of hNit2/?-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/?-amidase. A catalytic triad and the loop residues 116-128 of hNit2 play an essential role in supporting the stability of the enzyme-substrate complex, resulting in the generation of the catalytic products. These observations are predicted to be of benefit in the design of new inhibitors or activators for research involving cancer and hyperammonemic diseases.
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The role of glutamine synthetase and glutamate dehydrogenase in cerebral ammonia homeostasis.
Neurochem. Res.
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In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [(13)N]ammonia have shown that about 25 % of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of ?-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [(13)N]ammonia in rat brain is incorporated into glutamate and the ?-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/?-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/?-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: L-Aspartate + GTP + H(2)O ? Fumarate + GDP + P(i) + NH(3)) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research.
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Comparative enzymology of (2S,4R)4-fluoroglutamine and (2S,4R)4-fluoroglutamate.
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
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Many cancer cells have a strong requirement for glutamine. As an aid for understanding this phenomenon the (18)F-labeled 2S,4R stereoisomer of 4-fluoroglutamine [(2S,4R)4-FGln] was previously developed for in vivo positron emission tomography (PET). In the present work, comparative enzymological studies of unlabeled (2S,4R)4-FGln and its deamidated product (2S,4R)4-FGlu were conducted as an adjunct to these PET studies. Our findings are as follows: Rat kidney preparations catalyze the deamidation of (2S,4R)4-FGln. (2,4R)4-FGln and (2S,4R)4-FGlu are substrates of various aminotransferases. (2S,4R)4-FGlu is a substrate of glutamate dehydrogenase, but not of sheep brain glutamine synthetase. The compound is, however, a strong inhibitor of this enzyme. Rat liver cytosolic fractions catalyze a ?-elimination reaction with (2S,4R)4-FGlu, generating ?-ketoglutarate. Coupling of a deamidase reaction with this ?-elimination reaction provides an explanation for the previous detection of (18)F(-) in tumors exposed to [(18)F](2S,4R)4-FGln. One enzyme contributing to this reaction was identified as alanine aminotransferase, which catalyzes competing ?-elimination and aminotransferase reactions with (2S,4R)4-FGlu. This appears to be the first description of an aminotransferase catalyzing a ?-elimination reaction. The present results demonstrate that (2S,4R)4-FGln and (2S,4R)4-FGlu are useful analogues for comparative studies of various glutamine- and glutamate-utilizing enzymes in normal and cancerous mammalian tissues, and suggest that tumors may metabolize (2S,4R)4-FGln in a generally similar fashion to glutamine. In plants, yeast and bacteria a major route for ammonia assimilation involves the consecutive action of glutamate synthase plus glutamine synthetase (glutamate synthase cycle). It is suggested that (2S,4R)4-FGln and (2S,4R)4-FGlu will be useful probes in studies of ammonia assimilation by the glutamate synthase pathway in these organisms. Finally, glutamine transaminases are conserved in mammals, plants and bacteria, and probably serve to close the methionine salvage pathway, thus linking nitrogen metabolism to sulfur metabolism and one-carbon metabolism. It is suggested that (2S,4R)4-FGln may be useful in studies of the methionine salvage pathway in a variety of organisms.
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Core competencies for disaster medicine and public health.
Disaster Med Public Health Prep
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Effective preparedness, response, and recovery from disasters require a well-planned, integrated effort with experienced professionals who can apply specialized knowledge and skills in critical situations. While some professionals are trained for this, others may lack the critical knowledge and experience needed to effectively perform under stressful disaster conditions. A set of clear, concise, and precise training standards that may be used to ensure workforce competency in such situations has been developed. The competency set has been defined by a broad and diverse set of leaders in the field and like-minded professionals through a series of Web-based surveys and expert working group meetings. The results may provide a useful starting point for delineating expected competency levels of health professionals in disaster medicine and public health.
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?-Glutamylamines and neurodegenerative diseases.
Amino Acids
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Transglutaminases catalyze the formation of ?-glutamylamines utilizing glutamyl residues and amine-bearing compounds such as lysyl residues and polyamines. These ?-glutamylamines can be released from proteins by proteases in an intact form. The free ?-glutamylamines can be catabolized to 5-oxo-L-proline and the free amine by ?-glutamylamine cyclotransferase. Free ?-glutamylamines, however, accumulate in the CSF and affected areas of Huntington Disease brain. This observation suggests transglutaminase-derived ?-glutamylamines may play a more significant role in neurodegeneration than previously thought. The following monograph reviews the metabolism of ?-glutamylamines and examines the possibility that these species contribute to neurodegeneration.
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