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Find video protocols related to scientific articles indexed in Pubmed.
Adiponectin Induces A20 Expression in Adipose Tissue To Confer Metabolic Benefit.
Diabetes
PUBLISHED: 09-04-2014
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Obesity is a major risk factor for metabolic disease, with white adipose tissue (WAT) inflammation emerging as a key underlying pathology. We detail that mice lacking Reverb? exhibit enhanced fat storage without the predicted increased WAT inflammation or loss of insulin sensitivity. In contrast to most animal models of obesity and obese human patients, Reverb?(-/-) mice exhibit elevated serum adiponectin levels and increased adiponectin secretion from WAT explants in vitro, highlighting a potential anti-inflammatory role of this adipokine in hypertrophic WAT. Indeed, adiponectin was found to suppress primary macrophage responses to lipopolysaccharide and proinflammatory fatty acids, and this suppression depended on glycogen synthase kinase 3? activation and induction of A20. Attenuated inflammatory responses in Reverb?(-/-) WAT depots were associated with tonic elevation of A20 protein and ex vivo shown to depend on A20. We also demonstrate that adipose A20 expression in obese human subjects exhibits a negative correlation with measures of insulin sensitivity. Furthermore, bariatric surgery-induced weight loss was accompanied by enhanced WAT A20 expression, which is positively correlated with increased serum adiponectin and improved metabolic and inflammatory markers, including C-reactive protein. The findings identify A20 as a mediator of adiponectin anti-inflammatory action in WAT and a potential target for mitigating obesity-related pathology.
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Evidence that multiple defects in lipid regulation occur before hyperglycemia during the prodrome of type-2 diabetes.
PLoS ONE
PUBLISHED: 09-03-2014
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Blood-vessel dysfunction arises before overt hyperglycemia in type-2 diabetes (T2DM). We hypothesised that a metabolomic approach might identify metabolites/pathways perturbed in this pre-hyperglycemic phase. To test this hypothesis and for specific metabolite hypothesis generation, serum metabolic profiling was performed in young women at increased, intermediate and low risk of subsequent T2DM.
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The pathogenic mechanism of diabetes varies with the degree of overexpression and oligomerization of human amylin in the pancreatic islet ? cells.
FASEB J.
PUBLISHED: 08-19-2014
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The aggregation of human amylin (hA) to form cytotoxic structures has been closely associated with the causation of type 2 diabetes. We sought to advance understanding of how altered expression and aggregation of hA might link ?-cell degeneration with diabetes onset and progression, by comparing phenotypes between homozygous and hemizygous hA-transgenic mice. The homozygous mice displayed elevated islet hA that correlated positively with measures of oligomer formation (r=0.91; P<0.0001). They also developed hyperinsulinemia with transient insulin resistance during the prediabetes stage and then underwent rapid ?-cell loss, culminating in severe juvenile-onset diabetes. The prediabetes stage was prolonged in the hemizygous mice, wherein ?-cell dysfunction and extensive oligomer formation occurred in adulthood at a much later stage, when hA levels were lower (r=-0.60; P<0.0001). This is the first report to show that hA-evoked diabetes is associated with age, insulin resistance, progressive islet dysfunction, and ?-cell apoptosis, which interact variably to cause the different diabetes syndromes. The various levels of hA elevation cause different extents of oligomer formation in the disease stages, thus eliciting early- or adult-onset diabetes syndromes, reminiscent of type 1 and 2 diabetes, respectively. Thus, the hA-evoked diabetes phenotypes differ substantively according to degree of amylin overproduction. These findings are relevant to the understanding of the pathogenesis and the development of experimental therapeutics for diabetes.-Zhang, S., Liu, H., Chuang, C. L., Li, X., Au, M., Zhang, L., Phillips, A R. J., Scott, D. W., Cooper, G. J. S. The pathogenic mechanism of diabetes varies with the degree of overexpression and oligomerization of human amylin in the pancreatic islet ? cells.
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Diabetic cardiomyopathy is associated with defective myocellular copper regulation and both defects are rectified by divalent copper chelation.
Cardiovasc Diabetol
PUBLISHED: 04-10-2014
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Heart disease is the leading cause of death in diabetic patients, and defective copper metabolism may play important roles in the pathogenesis of diabetic cardiomyopathy (DCM). The present study sought to determine how myocardial copper status and key copper-proteins might become impaired by diabetes, and how they respond to treatment with the Cu (II)-selective chelator triethylenetetramine (TETA) in DCM.
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Essential roles of insulin, AMPK signaling and lysyl and prolyl hydroxylases in the biosynthesis and multimerization of adiponectin.
Mol. Cell. Endocrinol.
PUBLISHED: 04-09-2014
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Post-translational modifications (PTMs) of the adiponectin molecule are essential for its full bioactivity, and defects in PTMs leading to its defective production and multimerization have been linked to the mechanisms of insulin resistance, obesity, and type-2 diabetes. Here we observed that, in differentiated 3T3-L1 adipocytes, decreased insulin signaling caused by blocking of insulin receptors (InsR) with an anti-InsR blocking antibody, increased rates of adiponectin secretion, whereas concomitant elevations in insulin levels counteracted this effect. Adenosine monophosphate-activated protein kinase (AMPK) signaling regulated adiponectin production by modulating the expression of adiponectin receptors, the secretion of adiponectin, and eventually the expression of adiponectin itself. We found that lysyl hydroxylases (LHs) and prolyl hydroxylases (PHs) were expressed in white-adipose tissue of ob/ob mice, wherein LH3 levels were increased compared with controls. In differentiated 3T3-L1 adipocytes, both non-specific inhibition of LHs and PHs by dipyridyl, and specific inhibition of LHs by minoxidil and of P4H with ethyl-3,4-dihydroxybenzoate, caused significant suppression of adiponectin production, more particularly of the higher-order isoforms. Transient gene knock-down of LH3 (Plod3) caused a suppressive effect, especially on the high molecular-weight (HMW) isoforms. These data indicate that PHs and LHs are both required for physiological adiponectin production and in particular are essential for the formation/secretion of the HMW isoforms.
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?-Calcitonin gene related peptide (?-CGRP) mediated lipid mobilization in 3T3-L1 adipocytes.
Peptides
PUBLISHED: 03-24-2014
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The neuropeptide, ?-calcitonin gene-related peptide (?-CGRP), is expressed from sensory nerves that innervate fat. However, how ?-CGRP may act in adipose tissue is unclear. Using 3T3-L1 adipocytes we observed that rat ?-CGRP (r?-CGRP) evoked either a biphasic or monophasic reduction in intracellular free fatty acid (FFA) content. cAMP production was always monophasic and occurred when FFA responses were absent. Taken together with the observed potencies, these findings suggest that adipose tissue is a physiological target for ?-CGRP. However, uncoupling of the FFA and CGRP-signaling responses with increasing passage number limits 3T3-L1 adipocytes as a suitable cellular model.
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A label-free selected reaction monitoring workflow identifies a subset of pregnancy specific glycoproteins as potential predictive markers of early-onset pre-eclampsia.
Mol. Cell Proteomics
PUBLISHED: 07-29-2013
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Pre-eclampsia (PE) is a serious complication of pregnancy with potentially life threatening consequences for both mother and baby. Presently there is no test with the required performance to predict which healthy first-time mothers will go on to develop PE. The high specificity, sensitivity, and multiplexed nature of selected reaction monitoring holds great potential as a tool for the verification and validation of putative candidate biomarkersfor disease states. Realization of this potential involves establishing a high throughput, cost effective, reproducible sample preparation workflow. We have developed a semi-automated HPLC-based sample preparation workflow before a label-free selected reaction monitoring approach. This workflow has been applied to the search for novel predictive biomarkers for PE. To discover novel candidate biomarkers for PE, we used isobaric tagging to identify several potential biomarker proteins in plasma obtained at 15 weeks gestation from nulliparous women who later developed PE compared with pregnant women who remained healthy. Such a study generates a number of "candidate" biomarkers that require further testing in larger patient cohorts. As proof-of-principle, two of these proteins were taken forward for verification in a 100 women (58 PE, 42 controls) using label-free SRM. We obtained reproducible protein quantitation across the 100 samples and demonstrated significant changes in protein levels, even with as little as 20% change in protein concentration. The SRM data correlated with a commercial ELISA, suggesting that this is a robust workflow suitable for rapid, affordable, label-free verification of which candidate biomarkers should be taken forward for thorough investigation. A subset of pregnancy-specific glycoproteins (PSGs) had value as novel predictive markers for PE.
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Protection of the heart by treatment with a divalent-copper-selective chelator reveals a novel mechanism underlying cardiomyopathy in diabetic rats.
Cardiovasc Diabetol
PUBLISHED: 06-20-2013
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Intracellular calcium (Ca²?) coordinates the cardiac contraction cycle and is dysregulated in diabetic cardiomyopathy. Treatment with triethylenetetramine (TETA), a divalent-copper-selective chelator, improves cardiac structure and function in patients and rats with diabetic cardiomyopathy, but the molecular basis of this action is uncertain. Here, we used TETA to probe potential linkages between left-ventricular (LV) copper and Ca²? homeostasis, and cardiac function and structure in diabetic cardiomyopathy.
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Synthesis of the IGF-II-like hormone vesiculin using regioselective formation of disulfide bonds.
Org. Biomol. Chem.
PUBLISHED: 03-28-2013
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Diabetes mellitus, characterised by hyperglycemia and altered ?-cell function, is an increasingly common disorder affecting millions of individuals world-wide. While therapeutic regimens exist to manage the condition, diabetic individuals remain prone to complications that are detrimental to both their length and quality of life. An improved understanding of the disease which may then enable development of new treatments is therefore a desirable goal. Vesiculin, a novel IGF-II-like protein was recently isolated from the secretory granules of murine ?-cells, and preliminary studies indicate it is capable of signalling via the insulin receptor (IR)/insulin-like growth factor receptor 1(IGF1R) family giving it the potential to elicit both metabolic and mitogenic responses in the beta-cell. In order to facilitate further studies on this new member of the insulin-family of hormones, we undertook a chemical synthesis of the protein using regioselective disulfide bond formation.
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Synthesis of stable isotope-labelled monolysyl advanced glycation endproducts.
Amino Acids
PUBLISHED: 03-04-2013
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Advanced Glycation Endproducts (AGEs) are modified amino acids that form on proteins and are known to be implicated in the pathogenesis of diabetes and related diseases. Ready access to synthetic stable isotope-labelled AGEs allows for quantitative mass spectrometry studies to be undertaken, providing key insights into the roles AGEs play in the progression of such diseases. However, the majority of current syntheses of these compounds suffer from poor yields and lengthy procedures and are not suitable for the purposes required here. Here, we report robust syntheses of stable isotope-labelled monolysyl AGEs, N(?)-(carboxymethyl)lysine, N(?)-(carboxyethyl)lysine and pyrraline, that provide straightforward access to these compounds for quantitative amino acid analysis. This work will facilitate future investigations with these compounds and lead to a better understanding of the roles they play in diabetes and related diseases.
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Treatment with a copper-selective chelator causes substantive improvement in cardiac function of diabetic rats with left-ventricular impairment.
Cardiovasc Diabetol
PUBLISHED: 01-14-2013
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Defective copper regulation is implicated as a causative mechanism of organ damage in diabetes. Treatment with trientine, a divalent-copper-selective chelator, improves arterial and renal structure/function in diabetes, wherein it also ameliorates left-ventricular (LV) hypertrophy. However, direct in vivo evidence that trientine can improve cardiac function in heart failure has hitherto been lacking.
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A simple and rapid method for identifying and semi-quantifying peptide hormones in isolated pancreatic islets by direct-tissue matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Rapid Commun. Mass Spectrom.
PUBLISHED: 10-18-2011
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We describe a new, simple, robust and efficient method based on direct-tissue matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that enables consistent semi-quantitation of peptide hormones in isolated pancreatic islets from normal and diabetic rodents. Prominent signals were measured that corresponded to all the main peptide hormones present in islet-endocrine cells: (?-cells) glucagon, glicentin-related polypeptide/GRPP; (?-cells) insulin I, insulin II, C-peptide I, C-peptide II, amylin; (?-cells) somatostatin-14; and (PP-cells), and pancreatic polypeptide. The signal ratios coincided with known relative hormone abundances. The method demonstrated that severe insulin deficiency is accompanied by elevated levels of all non-?-cell-hormones in diabetic rat islets, consistent with alleviation of paracrine suppression of hormone production by non-?-cells. It was also effective in characterizing hormonal phenotype in hemizygous human-amylin transgenic mice that express human and mouse amylin in approx. equimolar quantities. Finally, the method demonstrated utility in basic peptide-hormone discovery by identifying a prominent new Gcg-gene-derived peptide (theoretical monoisotopic molecular weight 3263.5?Da), closely related to but distinct from GRPP, in diabetic islets. This peptide, whose sequence is HAPQDTEENARSFPASQTEPLEDPNQINE in Rattus norvegicus, could be a peptide hormone whose roles in physiology and metabolic disease warrant further investigation. This method provides a powerful new approach that could provide important new insights into the physiology and regulation of peptide hormones in islets and other endocrine tissues. It has potentially wide-ranging applications that encompass endocrinology, pharmacology, phenotypic analysis in genetic models of metabolic disease, and hormone discovery, and could also effectively limit the numbers of animals required for such studies.
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A unique case of neural amyloidoma diagnosed by mass spectrometry of formalin-fixed tissue using a novel preparative technique.
Amyloid
PUBLISHED: 08-24-2011
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We report here a unique amyloidoma of the radial nerve which could not be subtyped by available techniques, including immunohistochemistry and standard clinical and laboratory evaluation. In order to identify the amyloid monomer, we developed a novel preparative procedure designed to optimize conditions for liquid chromatography tandem mass spectrometry analysis of formalin-fixed/paraffin-embedded (FFPE) tissue. Subsequent mass spectrometric analysis clearly identified kappa light chain as the monomer, with no evidence of lambda light chain. Manual interpretation of the matched spectra revealed no evidence of polyclonality. This study also enabled detailed characterisation of twelve likely amyloid matrix components. Finally, our analysis revealed extensive hydroxylation of collagen type I but, unexpectedly, an almost complete lack of hydroxylated residues in the normally heavily-hydroxylated collagen type VI chains, pointing to structural/functional alterations of collagen VI in this matrix that could have contributed to the pathogenesis of this very unusual tumour. Given the high quality of the data here acquired using a standard quadrupole-time of flight tandem mass spectrometer of modest performance, the robust and straightforward preparative method described constitutes a competitive alternative to more involved approaches using state-of-the-art equipment.
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Therapeutic potential of copper chelation with triethylenetetramine in managing diabetes mellitus and Alzheimers disease.
Drugs
PUBLISHED: 07-21-2011
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This article reviews recent evidence, much of which has been generated by my groups research programme, which has identified for the first time a previously unknown copper-overload state that is central to the pathogenesis of diabetic organ damage. This state causes tissue damage in the blood vessels, heart, kidneys, retina and nerves through copper-mediated oxidative stress. This author now considers this copper-overload state to provide an important new target for therapeutic intervention, the objective of which is to prevent or reverse the diabetic complications. Triethylenetetramine (TETA) has recently been identified as the first in a new class of anti-diabetic molecules through the original work reviewed here, thus providing a new use for this molecule, which was previously approved by the US FDA in 1985 as a second-line treatment for Wilsons disease. TETA acts as a highly selective divalent copper (Cu(II)) chelator that prevents or reverses diabetic copper overload, thereby suppressing oxidative stress. TETA treatment of diabetic animals and patients has identified and quantified the interlinked defects in copper metabolism that characterize this systemic copper overload state. Copper overload in diabetes mellitus differs from that in Wilsons disease through differences in their respective causative molecular mechanisms, and resulting differences in tissue localization and behaviour of the excess copper. Elevated pathogenetic tissue binding of copper occurs in diabetes. It may well be mediated by advanced-glycation endproduct (AGE) modification of susceptible amino-acid residues in long-lived fibrous proteins, for example, connective tissue collagens in locations such as blood vessel walls. These AGE modifications can act as localized, fixed endogenous chelators that increase the chelatable-copper content of organs such as the heart and kidneys by binding excessive amounts of catalytically active Cu(II) in specific vascular beds, thereby focusing the related copper-mediated oxidative stress in susceptible tissues. In this review, summarized evidence from our clinical studies in healthy volunteers and diabetic patients with left-ventricular hypertrophy, and from nonclinical models of diabetic cardiac, arterial, renal and neural disease is used to construct descriptions of the mechanisms by which TETA treatment prevents injury and regenerates damaged organs. Our recent phase II proof-of-principle studies in patients with type 2 diabetes and in nonclinical models of diabetes have helped to define the pathogenetic defects in copper regulation, and have shown that they are reversible by TETA. The drug tightly binds and extracts excess systemic Cu(II) into the urine whilst neutralizing its catalytic activity, but does not cause systemic copper deficiency, even after prolonged use. Its physicochemical properties, which are pivotal for its safety and efficacy, clearly differentiate it from all other clinically available transition metal chelators, including D-penicillamine, ammonium tetrathiomolybdate and clioquinol. The studies reviewed here show that TETA treatment is generally effective in preventing or reversing diabetic organ damage, and support its ongoing development as a new medicine for diabetes. Trientine (TETA dihydrochloride) has been used since the mid-1980s as a second-line treatment for Wilsons disease, and our recent clinical studies have reinforced the impression that it is likely to be safe for long-term use in patients with diabetes and related metabolic disorders. There is substantive evidence to support the view that diabetes shares many pathogenetic mechanisms with Alzheimers disease and vascular dementia. Indeed, the close epidemiological and molecular linkages between them point to Alzheimers disease/vascular dementia as a further therapeutic target where experimental pharmacotherapy with TETA could well find further clinical application.
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Early organ-specific mitochondrial dysfunction of jejunum and lung found in rats with experimental acute pancreatitis.
HPB (Oxford)
PUBLISHED: 03-29-2011
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Multiple organ dysfunction is the main cause of death in severe acute pancreatitis. Primary mitochondrial dysfunction plays a central role in the development and progression of organ failure in critical illness. The present study investigated mitochondrial function in seven tissues during early experimental acute pancreatitis.
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Robust early pregnancy prediction of later preeclampsia using metabolomic biomarkers.
Hypertension
PUBLISHED: 09-15-2010
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Preeclampsia is a pregnancy-specific syndrome that causes substantial maternal and fetal morbidity and mortality. The etiology is incompletely understood, and there is no clinically useful screening test. Current metabolomic technologies have allowed the establishment of metabolic signatures of preeclampsia in early pregnancy. Here, a 2-phase discovery/validation metabolic profiling study was performed. In the discovery phase, a nested case-control study was designed, using samples obtained at 15±1 weeks gestation from 60 women who subsequently developed preeclampsia and 60 controls taking part in the prospective Screening for Pregnancy Endpoints cohort study. Controls were proportionally population matched for age, ethnicity, and body mass index at booking. Plasma samples were analyzed using ultra performance liquid chromatography-mass spectrometry. A multivariate predictive model combining 14 metabolites gave an odds ratio for developing preeclampsia of 36 (95% CI: 12 to 108), with an area under the receiver operator characteristic curve of 0.94. These findings were then validated using an independent case-control study on plasma obtained at 15±1 weeks from 39 women who subsequently developed preeclampsia and 40 similarly matched controls from a participating center in a different country. The same 14 metabolites produced an odds ratio of 23 (95% CI: 7 to 73) with an area under receiver operator characteristic curve of 0.92. The finding of a consistent discriminatory metabolite signature in early pregnancy plasma preceding the onset of preeclampsia offers insight into disease pathogenesis and offers the tantalizing promise of a robust presymptomatic screening test.
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The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP suppress misfolding and formation of ?-sheet-containing aggregates by human amylin: a potential role for defective chaperone biology in Type 2 diabetes.
Biochem. J.
PUBLISHED: 08-26-2010
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Misfolding of the islet ?-cell peptide hA (human amylin) into ?-sheet-containing oligomers is linked to ?-cell apoptosis and the pathogenesis of T2DM (Type 2 diabetes mellitus). In the present study, we have investigated the possible effects on hA misfolding of the chaperones HSP (heat-shock protein) 70, GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) and HSP40/DnaJ. We demonstrate that hA underwent spontaneous time-dependent ?-sheet formation and aggregation by thioflavin-T fluorescence in solution, whereas rA (rat amylin) did not. HSP70, GRP78/BiP and HSP40/DnaJ each independently suppressed hA misfolding. Maximal molar protein/hA ratios at which chaperone activity was detected were 1:200 (HSP70, HSP40/DnaJ and GRP78/BiP). By contrast, none of the chaperones modified the secondary structure of rA. hA, but not rA, was co-precipitated independently with HSP70 and GRP78/BiP by anti-amylin antibodies. As these effects occur at molar ratios consistent with chaperone binding to relatively rare misfolded hA species, we conclude that HSP70 and GRP78/BiP can detect and bind misfolded hA oligomers, thereby effectively protecting hA against bulk misfolding and irreversible aggregation. Defective ?-cell chaperone biology could contribute to hA misfolding and initiation of apoptosis in T2DM.
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Illuminating the molecular basis of diabetic arteriopathy: a proteomic comparison of aortic tissue from diabetic and healthy rats.
Proteomics
PUBLISHED: 08-14-2010
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Arterial disease is a major diabetic complication, yet the component molecular mechanisms of diabetic arteriopathy remain poorly understood. In order to identify major proteins/pathways implicated in diabetic arteriopathy, we studied the effect of 16-wk untreated streptozotocin-induced diabetes on the rat aortic proteome. Specific protein levels in isolated aortas were compared in six discrete, pair-wise (streptozotocin-diabetic and non-diabetic age-matched controls) experiments in which individual proteins were identified and quantified by iTRAQ combined with LC-MS/MS. A total of 398 unique non-redundant proteins were identified in at least one experiment and 208 were detected in three or more. Between-group comparisons revealed significant changes or trends towards changes in relative abundance of 51 proteins (25 increased, 26 decreased). Differences in levels of selected proteins were supported by Western blotting and/or enzyme assays. The most prominent diabetes-associated changes were in groups of proteins linked to oxidative stress responses and the structure/function of myofibrils and microfilaments. Indexes of mitochondrial content were measurably lower in aortic tissue from diabetic animals. Functional cluster analysis also showed decreased levels of glycolytic enzymes and mitochondrial electron transport system-complex components. These findings newly implicate several proteins/functional pathways in the pathogenesis of arteriosclerosis/diabetic arteriopathy.
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Mice lacking the neuropeptide alpha-calcitonin gene-related peptide are protected against diet-induced obesity.
Endocrinology
PUBLISHED: 07-07-2010
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Alpha-calcitonin gene-related peptide (alphaCGRP) is a neuropeptide that is expressed in motor and sensory neurons. It is a powerful vasodilator and has been implicated in diverse metabolic roles. However, its precise physiological function remains unclear. In this study, we investigated the role of alphaCGRP in lipid metabolism by chronically challenging alphaCGRP-specific knockout (alphaCGRP(-/-)) and control mice with high-fat diet regimens. At the start of the study, both animal groups displayed similar body weights, serum lipid markers, and insulin sensitivity. However, alphaCGRP(-/-) mice displayed higher core temperatures, increased energy expenditures, and a relative daytime (nonactive) depression in respiratory quotients, which indicated increased beta-oxidation. In response to fat feeding, alphaCGRP(-/-) mice were comparatively protected against diet-induced obesity with an attenuated body weight gain and an overall reduction in adiposity across all the three diets examined. AlphaCGRP(-/-) mice also displayed improved glucose handling and insulin sensitivity, lower im and hepatic lipid accumulation, and improved overall metabolic health. These findings define a new role for alphaCGRP as a mediator of energy metabolism and opens up therapeutic opportunities to target CGRP action in obesity.
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Changes in the mesenteric lymph proteome induced by hemorrhagic shock.
Shock
PUBLISHED: 02-18-2010
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Biologically active factors produced by the intestine and transported by the aqueous and protein fraction of mesenteric lymph are now thought to contribute significantly to the development of distant organ failure in hemorrhagic shock. Despite the likely relevance of the protein composition of mesenteric lymph conditioned by hemorrhagic shock, there is no detailed description of its proteome. The aim of this study was to provide the first comprehensive description of the proteome of hemorrhagic shock-conditioned mesenteric lymph. Mesenteric lymph was collected from 16 male Wistar rats randomized to group 1 (n = 8) sham control and group 2 (n = 8) with hemorrhagic shock. The lymph was subjected to proteomic analysis using iTRAQ and liquid chromatography-tandem mass spectrometry. Sixty of the 245 proteins had a significant increase in their relative abundance in the hemorrhagic shock group. A bioinformatics approach highlighted the importance of the key gene ontology pathways relating to response to injury and metabolic responses as changing most significantly in shock. Using an interactome, we identified several highly connected proteins: 14-3-3 Zeta, 14-3-3 epsilon, actin, aldolase A, calmodulin, cofilin 1, cystatin C, fatty acid-binding protein 4, profilin 1, prolyl 4-hydrolase, peptidylprolyl isomerase, and transgelin. This study provides the first detailed description of protein changes in hemorrhagic shock-conditioned mesenteric lymph, and using a bioinformatics approach, we identified several targets for possible further research.
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Pharmacokinetics, pharmacodynamics, and metabolism of triethylenetetramine in healthy human participants: an open-label trial.
J Clin Pharmacol
PUBLISHED: 02-09-2010
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The selective Cu(II)-chelator, triethylenetetramine (TETA), is undergoing clinical trials for the treatment of heart failure in patients with diabetes. Recently, the authors showed that 2 acetylated metabolites, N(1)-acetyltriethylenetetramine (MAT) and N(1),N(10)-diacetyltriethylenetetramine (DAT), are formed in humans following oral TETA administration. Thus, it became necessary to determine whether the N-acetyltransferase (NAT) 2 phenotype has any effects on the pharmacological properties and safety profile of TETA. Twelve fast and 12 slow NAT2-phenotype healthy participants were recruited. After oral drug administration, the authors collected plasma and urine samples, measured plasma concentrations of TETA and its 2 metabolites along with concomitant urinary copper concentrations, and performed safety tests. They present, for the first time, the complete 24-hour pharmacokinetic profiles of TETA, MAT, and DAT in humans. There was no evidence for clear-cut differences in pharmacokinetic profiles between fast and slow acetylators. Pharmacodynamic analysis showed no significant differences in cupruresis between the 2 NAT2 phenotypes. Safety results were consistent with TETA being well tolerated, and no significant differences in safety profiles were observed between the 2 phenotypes. Based on these data, NAT2 phenotype does not affect TETAs pharmacokinetic, pharmacodynamic, or safety profiles. TETA may be acetylated via an alternative mechanism, such as that catalyzed by spermidine/spermine N(1)-acetyltranferase.
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A novel two-chain IGF-II-derived peptide from purified ?-cell granules.
Growth Horm. IGF Res.
PUBLISHED: 01-26-2010
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Insulin-like growth factor II (IGF-II) is a potent mitogen that regulates prenatal growth and development in both humans and rodents. Its role in post-natal life is less clear although immunohistochemical studies have observed IGF-II-like immunoreactivity (IGF-II-LI) associated with insulin-producing pancreatic ?-cells. Here we isolated secretory granules from a ?-cell line, ?TC6-F7, and characterized the nature of the IGF-II-LI located therein.
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Tetracycline treatment retards the onset and slows the progression of diabetes in human amylin/islet amyloid polypeptide transgenic mice.
Diabetes
PUBLISHED: 09-30-2009
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Aggregation of human amylin/islet amyloid polypeptide (hA/hIAPP) into small soluble beta-sheet-containing oligomers is linked to islet beta-cell degeneration and the pathogenesis of type 2 diabetes. Here, we used tetracycline, which modifies hA/hIAPP oligomerization, to probe mechanisms whereby hA/hIAPP causes diabetes in hemizygous hA/hIAPP-transgenic mice.
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Aberrant processing of plasma vitronectin and high-molecular-weight kininogen precedes the onset of preeclampsia.
Reprod Sci
PUBLISHED: 08-05-2009
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To date, there is no reliable test to identify women in early pregnancy at risk of developing preeclampsia. Difference gel electrophoresis (DIGE) identified the plasma proteins vitronectin (VN) and high-molecular-weight kininogen (HK) in association with preeclampsia. In a longitudinal proteomics study, the plasma of preeclamptic patients (n = 6) was compared to healthy control participants (n = 6) before the onset of preeclampsia (week 20) and at the time of presentation with clinical disease (weeks 33-36). The 75-kd single-chain VN molecule increased 1.6- to 1.9-fold in preeclampsia, whereas the 65-kd moiety of the 2-chain VN molecule decreased 1.5- to 1.7-fold compared to healthy controls (P < .05). Immunoblots revealed differences in proteolytic processing of VN and/or HK in women who develop preeclampsia or preeclampsia further complicated by small-for-gestational-age. Vitronectin and HK may prove to be useful as early markers of fibrinolytic activity and neutrophil activation, which are known to be associated with preeclampsia.
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Quantitative proteomic profiling identifies new renal targets of copper(II)-selective chelation in the reversal of diabetic nephropathy in rats.
Proteomics
PUBLISHED: 07-28-2009
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This study aimed to identify new diabetic nephropathy (DN)-related proteins and renal targets of the copper(II)-selective chelator, triethylenetetramine (TETA) in streptozotocin-diabetic rats. We used the recently developed iTRAQ technology to compare renal protein profiles among non-diabetic, diabetic, and TETA-treated diabetic rats. In diabetic kidneys, tubulointerstitial nephritis antigen (TINag), voltage-dependent anion-selective channel (VDAC) 1, and VDAC2 were up-regulated in parallel with alterations in expression of proteins with functions in oxidative stress and oxidative phosphorylation (OxPhos) pathways. By contrast, mitochondrial HSP 60, Cu/Zn-superoxide dismutase, glutathione S-transferase alpha3 and aquaporin-1 were down-regulated in diabetic kidneys. Following TETA treatment, levels of D-amino acid oxidase-1, epoxide hydrolase-1, aquaporin-1, and a number of mitochondrial proteins were normalized, with concomitant amelioration of albuminuria. Changes in levels of TINag, collagen VIalpha1, actinin 4alpha, apoptosis-inducing factor 1, cytochrome C, histone H3, VDAC1, and aquaporin-1 were confirmed by Western blotting or immunohistochemistry. Changes in expression of proteins related to tubulointerstitial function, podocyte structure, and mitochondrial apoptosis are implicated in the mechanism of DN and their reversal by TETA. These findings are consistent with the hypothesis that this new experimental therapy may be useful for treatment of DN.
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Pharmacokinetic and pharmacodynamic modeling of a copper-selective chelator (TETA) in healthy adults.
J Clin Pharmacol
PUBLISHED: 07-16-2009
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The population pharmacokinetics (PK) and pharmacodynamics (PD) of triethylenetetramine (TETA) dihydrochloride (trientine, GC811007) administered orally as 100-, 300-, 600-, or 1800-mg twice-daily doses were assessed in healthy adult male and female volunteers. This study was a randomized, double-blind, placebo-controlled, group-sequential, dose-escalating design. Forty participants, 10 per dose level (8 receiving TETA, 2 receiving placebo), received twice-daily doses for 14 consecutive days. A 2-compartment model for the PK and a linear direct effect model for drug-induced copper excretion (PD) were employed. The population PK/PD model was applied using the NONMEM software. Covariates tested were glomerular filtration rate (GFR), body weight, and gender. Multiple daily doses of TETA were safe and generally well tolerated. The linear 2-compartment model with first-order absorption well characterized the serum concentration data. Although its role was small, GFR had a statistically significant (P < .05) influence on systemic clearance (CL/F). The augmentation of copper excretion was well described by a direct linear model in which the slope was related to GFR and gender (P < .001). The intersubject coefficient of variation was 22.2% for slope (SL) and 82.5% for intercept (ER0). TETA has consistent single/multiple-dose pharmacokinetics and dose-proportional and serum concentration-proportional effects on enhancing copper excretion.
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Impaired ATP turnover and ADP supply depress cardiac mitochondrial respiration and elevate superoxide in nonfailing spontaneously hypertensive rat hearts.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 06-24-2009
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Although most attention has been focused on mitochondrial ATP production and transfer in failing hearts, less has been focused on the nonfailing hypertensive heart. Here, energetic complications are less obvious, yet they may provide insight into disease ontogeny. We studied hearts from 12-mo-old spontaneously hypertensive rats (SHR) relative to normotensive Wistar-Kyoto (WKY) rats. The ex vivo working-heart model of SHR showed reduced compliance and impaired responses to increasing preloads. High-resolution respirometry showed higher state 3 (with excess ADP) respiration in SHR left ventricle fibers with complex I substrates and maximal uncoupled respiration with complex I + complex II substrates. Respiration with ATP was depressed 15% in SHR fibers relative to WKY fibers, suggesting impaired ATP hydrolysis. This finding was consistent with a 50% depression of actomyosin ATPase activities. Superoxide production from SHR fibers was similar to that from WKY fibers respiring with ADP; however, it was increased by 15% with ATP. In addition, the apparent K(m) for ADP was 54% higher for SHR fibers, and assays conducted after ex vivo work showed a 28% depression of complex I in SHR, but not WKY, fibers. Transmission electron microscopy showed similar mitochondrial volumes but a decrease in the number of cristae in SHR mitochondria. Tissue lipid peroxidation was also 15% greater in SHR left ventricle. Overall, these data suggest that although cardiac mitochondria from nonfailing SHR hearts function marginally better than those from WKY hearts, they show dysfunction after intense work. Impaired ATP turnover in hard-working SHR hearts may starve cardiac mitochondria of ADP and elevate superoxide.
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A proteomic approach identifies early pregnancy biomarkers for preeclampsia: novel linkages between a predisposition to preeclampsia and cardiovascular disease.
Proteomics
PUBLISHED: 05-16-2009
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Preeclampsia (PE) is a common, potentially life-threatening pregnancy syndrome triggered by placental factors released into the maternal circulation, resulting in maternal vascular dysfunction along with activated inflammation and coagulation. Currently there is no screening test for PE. We sought to identify differentially expressed plasma proteins in women who subsequently develop PE that may perform as predictive biomarkers. In seven DIGE experiments, we compared the plasma proteome at 20 wk gestation in women who later developed PE with an appropriate birth weight for gestational age baby (n=27) or a small for gestational age baby (n=12) to healthy controls with uncomplicated pregnancies (n=57). Of the 49 differentially expressed spots associated with PE-appropriate for gestational age, PE-small for gestational age or both (p<0.05, false discovery rate corrected), 39 were identified by LC-MS/MS. Two protein clusters that accurately (>90%) classified women at risk of developing PE were identified. Immunoblots confirmed the overexpression of fibrinogen gamma chain and alpha-1-antichymotrypsin in plasma prior to PE. The proteins identified are involved in lipid metabolism, coagulation, complement regulation, extracellular matrix remodeling, protease inhibitor activity and acute-phase responses, indicating novel synergism between pathways involved in the pathogenesis of PE. Our findings are remarkably similar to recently identified proteins complexed to high-density lipoprotein and linked to cardiovascular disease.
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An altered pattern of circulating apolipoprotein E3 isoforms is implicated in preeclampsia.
J. Lipid Res.
PUBLISHED: 04-03-2009
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Preeclampsia is a common pregnancy complication that is an important cause of preterm birth and fetal growth restriction. Because there is no diagnostic test yet available for preeclampsia, we used a proteomic approach to identify novel serum/plasma biomarkers for this condition. We conducted case control studies comparing nulliparous women who developed preeclampsia at 36-38 weeks of gestation with healthy nulliparous women matched by gestational age at sampling. Serum/plasma was depleted of six abundant proteins and analyzed by two-dimensional gel electrophoresis (n = 12 per group) and difference gel electrophoresis (n = 12 per group). Differences in abundance of protein spots were detected by univariate and multivariate statistical analyses. Proteins were identified by mass spectrometry and expression of selected proteins was validated by immunoblotting. Proteins whose concentrations were selectively associated with preeclampsia included apolipoprotein E (apoE), apoC-II, complement factor C3c, fibrinogen, transthyretin, and complement factor H-related protein 2. An increase in a deglycosylated isoform of apoE3 and concomitantly decreased amounts of one apoE3 glycoisoform were identified in preeclamptic plasma and confirmed by immunoblotting. Altered production of these preeclampsia-related apoE3 isoforms might impair reverse cholesterol transport, contributing to arterial damage. These findings point to a novel mechanistic link between preeclampsia and subsequent cardiovascular disease.
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The proteome of mesenteric lymph during acute pancreatitis and implications for treatment.
JOP
PUBLISHED: 03-17-2009
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The protein fraction of mesenteric lymph during acute pancreatitis and other critical illness is thought to contain toxic factors. However, we do not have a complete description of the mesenteric lymph proteome during acute pancreatitis.
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Proteins associated with immunopurified granules from a model pancreatic islet beta-cell system: proteomic snapshot of an endocrine secretory granule.
J. Proteome Res.
PUBLISHED: 02-28-2009
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beta-Cell granules contain proteins involved in fuel regulation, which when altered, contribute to metabolic disorders including diabetes mellitus. We analyzed proteins present in purified granules from the INS-1E beta-cell model. Fifty-one component proteins were identified by LC-MS/MS including hormones, granins, protein processing components, cellular trafficking components, enzymes implicated in cellular metabolism and chaperone proteins. These findings may increase understanding of granule secretion and the processes leading to protein aggregation and beta-cell death in type-2 diabetes.
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Adiponectin haploinsufficiency promotes mammary tumor development in MMTV-PyVT mice by modulation of phosphatase and tensin homolog activities.
PLoS ONE
PUBLISHED: 02-20-2009
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Adiponectin is an adipokine possessing beneficial effects on obesity-related medical complications. A negative association of adiponectin levels with breast cancer development has been demonstrated. However, the precise role of adiponectin deficiency in mammary carcinogenesis remains elusive.
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Complex formation equilibria of Cu(II) and Zn(II) with triethylenetetramine and its mono- and di-acetyl metabolites.
Dalton Trans
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Triethylenetetramine (TETA) dihydrochloride, or trientine, is a therapeutic molecule that has long been used as a copper-chelating agent for the second-line treatment of patients with Wilsons disease. More recently, it has also been employed as an experimental therapeutic molecule in diabetes where it improves cardiac structure in patients with diabetic cardiomyopathy and left-ventricular hypertrophy. TETA is metabolized by acetylation, which leads to the formation of two main metabolites in humans and other mammals, monoacetyl-TETA (MAT) and diacetyl-TETA (DAT). These metabolites have been identified in the plasma and urine of healthy and diabetic subjects treated with TETA, and could themselves play a role in TETA-mediated copper chelation and restoration of physiological copper regulation in diabetes. In this regard, a potentiometric and spectrophotometric study of Cu(II)-complex formation equilibria of TETA, MAT and DAT is presented here, to provide a comprehensive evaluation of the stoichiometries of the complexes formed and of their relative stability constants. A potentiometric study has also been conducted on the corresponding Zn(II) complexes, to evaluate any possible interference with TETA-mediated Cu(II) binding by this second physiological transition-metal ion, which is present in similar concentrations in human plasma and which also binds to TETA. An ESI-MS study of these systems has both confirmed the complex formation mechanisms established from the potentiometric and spectrophotometric results, and in addition provided direct information on the stoichiometry of the complexes formed in solution. These data when taken together show that the 1 : 1 complexes formed with Cu(II) and Zn(II) have different degrees of protonation. The stability of the Cu(II) and Zn(II) complexes with the three ligands, evaluated by the parameters pCu and pZn, decreases with the introduction of the acetyl groups. Nevertheless the stability of Cu(II) complexes with MAT is sufficiently high to enable its participation in copper scavenging from the patient. A speciation study of the behavior of TETA and MAT with Cu(II) in the presence of Zn(II) at peri-physiological plasma concentrations is also presented. While Zn(II) did not hinder copper binding, the possibility is raised that prolonged TETA treatment could possibly alter the homeostatic regulation of this essential metal ion. The lack of reliable literature stability constants concerning the Cu(II) and Zn(II) interaction with the major transport proteins in plasma is also briefly considered.
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Synthesis of monolysyl advanced glycation endproducts and their incorporation into collagen model peptides.
Org. Lett.
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The synthesis of advanced glycation endproducts (AGEs), CML, CEL, and pyrraline and their incorporation into collagen model peptides is reported. AGEs are modified amino acids that form on proteins such as collagen and are thought to play a significant role in the pathogenesis of many diseases, particularly diabetes. The synthesis and incorporation of these compounds into synthetic peptides is a key step in developing model systems with which to investigate AGE-modified proteins.
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Metabolomic analysis of rat serum in streptozotocin-induced diabetes and after treatment with oral triethylenetetramine (TETA).
Genome Med
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The prevalence, and associated healthcare burden, of diabetes mellitus is increasing worldwide. Mortality and morbidity are associated with diabetic complications in multiple organs and tissues, including the eye, kidney and cardiovascular system, and new therapeutics to treat these complications are required urgently. Triethylenetetramine (TETA) is one such experimental therapeutic that acts to chelate excess copper (II) in diabetic tissues and reduce oxidative stress and cellular damage.
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Plasma clusterin increased prior to small for gestational age (SGA) associated with preeclampsia and decreased prior to SGA in normotensive pregnancies.
Reprod Sci
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In our search for early biomarkers for the pregnancy complicationssmall for gestational age (SGA) and preeclampsia (PE) we analysed plasma from 19-21 weeks gestation in women recruited into the SCOPE study, a prospective cohort of nulliparous women, by differential in gel electrophoresis (DIGE). DIGE revealed the differential expression of clusterin levels and its isoforms in top6-depleted plasma of women who delivered an SGA infant but remained normotensive (SGA-NT; N = 8) compared to healthy women with an uncomplicated pregnancy outcome (Controls, N = 8). Immunosorbent enzyme-linked assay (ELISA) showed that compared to plasma clusterin levels from healthy controls [71.1 (SD 12.4) µg/mL, n = 39], clusterin was decreased in SGA-NT [58.3 (SD 11.7), N = 20, P < 0.0001], increased in women with SGA and PE [81.5 (SD 14.8), N = 20, P < 0.01], but similar in PE alone [71.2 (SD 9.4)g/ml, P = 1.0]. Screening for clusterin levels and/or its different isoformsmay be useful in mid-pregnancy to identify women who subsequently develop SGA but remain normotensive or who develop preeclampsia with SGA.
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3,12-Diaza-6,9-diazo-nia-2,13-dioxotetra-decane bis-(perchlorate).
Acta Crystallogr Sect E Struct Rep Online
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The crystal structure of the title diprotonated diacetyl-triethyl-ene-tetra-mine (DAT) perchorate salt, C(10)H(24)N(4)O(2) (2+)·2ClO(4) (-), can be described as a three-dimensional assembly of alternating layers consisting of diprotonated diacetyl-triethyl-ene-tetra-mine (H(2)DAT)(2+) strands along [100] and the anionic species ClO(4) (-). The (H(2)DAT)(2+) cations in the strands are connected via N-H?O hydrogen bonding between the acetyl groups and the amine groups of neighbouring (H(2)DAT)(2+) cations. Layers of (H(2)DAT)(2+) strands and perchlorate anions are connected by a network of hydrogen bonds between the NH and NH(2) groups and the O atoms of the perchlorate anion. The asymmetric unit consits of one perchlorate anion in a general position, as well as of one cation that is located on a center of inversion.
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Synthesis of glycosylated 5-hydroxylysine, an important amino acid present in collagen-like proteins such as adiponectin.
Org. Biomol. Chem.
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The synthesis of naturally occurring glycosylated (2S,5R)-hydroxylysine still remains a challenge. This perspective highlights the importance of this post-translationally modified amino acid residue in the observed bioactivity of collagen and related collagen-like proteins such as adiponectin, an important target for the treatment of type II diabetes. Strategies employed to date for the syntheses of (2S,5R)-hydroxylysine and the methods to effect glycosylation of this modified amino acid are also summarized herein.
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