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Impaired lipid metabolism is a common risk factor underlying several metabolic diseases such as metabolic syndrome and type 2 diabetes. Branched-chain amino acids (BCAAs) that include valine, leucine and isoleucine have been proven to share a role in lipid metabolism and hence in maintaining metabolic health. We have previously introduced a hypothesis suggesting that BCAA degradation mechanistically connects to lipid oxidation and storage in skeletal muscle. To test our hypothesis, the present study examined the effects of BCAA deprivation and supplementation on lipid oxidation, lipogenesis and lipid droplet characteristics in murine C2C12 myotubes. In addition, the role of myotube contractions on cell metabolism was studied by utilizing in vitro skeletal-muscle-specific exercise-like electrical pulse stimulation (EPS). Our results showed that the deprivation of BCAAs decreased both lipid oxidation and lipogenesis in C2C12 myotubes. BCAA deprivation further diminished the number of lipid droplets in the EPS-treated myotubes. EPS decreased lipid oxidation especially when combined with high BCAA supplementation. Similar to BCAA deprivation, high BCAA supplementation also decreased lipid oxidation. The present results highlight the role of an adequate level of BCAAs in healthy lipid metabolism.

Sulfomycins are sulfur-rich, ribosomally synthesized, and post-translationally modified peptides (RiPPs) that are characterized by a 35-membered macrocyclic ring system with a pyridine domain central to five azoles and additional dehydroamino acids. The pathway through which these large thiopeptide antibiotics are formed in

Elevation of branched-chain amino acids (BCAAs) in biological fluids indicates a deficiency in the branched-chain ketoacid dehydrogenase complex, which causes maple syrup urine disease (MSUD). Detection of increased levels of alloisoleucine confirms the diagnosis, while routine monitoring of leucine concentration is crucial for preventing metabolic decompensation and neurological dysfunction. In the metabolic center at Universidad de Chile, we have confirmed and monitored more than fifty MSUD patients in the last 20 years. Most diagnoses were made by clinical and sibling diagnosis, as MSUD is not included in the Chilean national newborn screening program. Shortening diagnosis time has a fundamental impact on the outcome of patients, therefore we focused on implementing detection of BCAAs in dried blood spot by liquid chromatography mass spectrometry (LC-MSMS) for disease confirmation as well as for biochemical monitoring. Retrospective analysis of samples from 9 diagnosed MSUD patients were performed; BCAAs values were determined via MSMS and LC-MSMS conducted in parallel. Leucine and alloisoleucine levels were positively correlated with patient’s diagnosis age. Alloisoleucine was significatively elevated as early as 24 hr after birth. A predictable variation in BCAAs levels after nutritional intervention among diagnosed MSUD patients was found.

DHTKD1 is the E1 component of the 2-oxoadipic acid dehydrogenase complex (OADHc), which functions in the L-lysine degradation pathway. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q (CMT2Q) and eosinophilic esophagitis (EoE). A crystal structure and inhibitors of DHTKD1 could improve the understanding of these clinically distinct disorders, but are currently not available. Here we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.1 Å. The protein assembles as a dimer with residues from both monomers contributing to cofactor binding. We also report the impact of ten DHTKD1 missense mutations on the encoded proteins by enzyme kinetics, thermal stability and structural modeling. Some DHTKD1 variants displayed impaired folding (S777P and S862I), whereas other substitutions rendered the enzyme inactive (L234G, R715C and R455Q) or affected the thermal stability and catalytic efficiency (V360A and P773L). Three variants (R163Q, Q305H and G729R) surprisingly showed wild type like properties. Our work provides a structural basis for further understanding of the function of DHTKD1 and a starting point for selective small molecule inhibitors of the enzyme, which could help tease apart the role of this enzyme in several human pathologies.

Background: Circulating branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) have been shown to be associated with insulin resistance and diabetes risk. The common rs1440581 T allele in the protein phosphatase Mg2+/Mn2+dependent 1K (PPM1K) gene has been related to elevated BCAA concentrations and risk of type 2 diabetes.Objective: In the present study, we tested whether dietary fat and carbohydrate intakes influenced the association between the rs1440581 PPM1K genetic variant and glucose-metabolism traits during weight loss.Design: The rs1440581 PPM1K genetic variant was genotyped in a total of 757 nondiabetic individuals who were randomly assigned to 1 of 2 energy-restricted diets that differed in macronutrient composition (low-fat diet: 20-25% fat, 15% protein, and 60-65% carbohydrate; high-fat diet: 40-45% fat, 15% protein, and 40-45% carbohydrate). The changes in fasting glucose, fasting insulin, insulin resistance (homeostasis model assessment of insulin resistance) and homeostasis model assessment of beta cell function (HOMA-B) were measured after a mean +/- SD weight loss of 6.8 +/- 3.4 kg over 10 wk and analyzed according to the presence of the T allele of rs1440581.Results: The rs1440581 T allele was associated with a smaller improvement in glucose concentrations after the 10-wk dietary intervention (beta +/- SE: 0.05 +/-.02 mg/dL; P = 0.03). In addition, significant gene-diet interactions were shown for the rs1440581 PPM1K genetic variant in relation to changes in insulin and HOMA-B (P-interaction = 0.006 and 0.002, respectively). In response to the high-fat diet, the T allele was associated with a higher reduction of insulin (beta +/- SE: -0.77 +/- 0.40 mu U/mL; P = 0.04) and HOMA-B (beta +/- SE: -13.2 +/- 3.81; P = 0.003). An opposite effect was observed in the low-fat diet group, although in this group the T allele was marginally (P = 0.10) and not significantly (P = 0.24) associated with insulin and HOMA-B, respectively.Conclusion: PPM1K rs1440581 may affect changes in glucose metabolism during weight loss, and this effect is dependent on dietary fat and carbohydrate intakes. This trial was registered at controlled-trials. com as ISRCTN25867281.

Sulfomycins are sulfur-rich, ribosomally synthesized, and post-translationally modified peptides (RiPPs) that are characterized by a 35-membered macrocyclic ring system with a pyridine domain centr...

Aim. The investigation of the influence of some physicochemical factors on dehydrogenase activity of Bacillus subtilis IMB B-7023. Methods. The bacteria were cultivated in the periodic process. Dehydrogenase activity was evaluated on reduction of 2,3,5-tetrazolium chloride to triphenilphormazane, the concentration of which was determined by photocolorymetrical methods. Results. It was defined that dehydrogenase activity reached the greatest significance in the given strain at the temperature of 37 °C in the media having pH 7.0. The highest indices of dehydrogenase activity have been obtained in the content of 10 g/l glucose in it. It was determined that ions of Mg2+ and Ca2+ have stimulated dehydrogenase activity of Bacillus subtilis, and ions of Fe3+, on the contrary, suppressed it. Сonclusion. Actual presence of high sensitiveness of the dehydrogenase complex of these bacteria to physicochemical factors of the environment can be used for making up the prognoses of the given strain activity under certain factors of the environment in its use as a complex bacterial preparation in agroecosystems.

Maple syrup urine disease (MSUD) and further cases were identified in herd mates of a small Hereford herd in Indiana based on history, clinical signs, microscopic lesions, and biochemical and genetic testing. This aminoacidopathy has been diagnosed in polled Shorthorn, polled Hereford, and Hereford cattle in Australia, Uruguay, Argentina, and Canada and is the result of a mutation of the branched-chain alpha-ketoacid dehydrogenase complex. The Indiana index calf case was confirmed by showing the classic accumulation of ketoacids in liver that results from a defect in the E1-alpha subunit (248 C/T haplotype) in the mitochondrial branched-chainα-ketoacid dehydrogenase complex. The presence of the mutation was confirmed in the index case, the dam, and four related herd mates that represent the first confirmed cases of bovine MSUD mutation in United States cattle.

We describe a male neonate with classic maple syrup urine disease (MSUD) in metabolic crisis. On day 7 of life, he was referred to hospital because of coma and metabolic acidosis with maple syrup odor. On day 4 after admission, brain magnetic resonance imaging findings were consistent with encephalopathy due to MSUD. Proton magnetic resonance spectroscopy ((1) H-MRS) showed a large methyl resonance peak at 0.9 p.p.m. The diagnosis of MSUD was confirmed on low branched-chain α-keto acid dehydrogenase complex activity in lymphocyte. (1) H-MR spectra were obtained in 10 min, while it took at least several days to obtain the results of other diagnostic examinations. In convalescence, the peak at 0.9 p.p.m. decreased. The large methyl resonance peak at 0.9 p.p.m. in brain (1) H-MRS would be one of the earliest clues to the diagnosis of classic MSUD in the neonatal period, especially in metabolic crisis.

The activity of mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC) is severely reduced in human pathologies where oxidative stress is traditionally thought to play an important role, such as familial and sporadic forms of Alzheimer's disease and other age-related neurodegenerative diseases. This minireview is focused on substantial data that were accumulated over the last 2 decades to support the concept that KGDHC can be a primary mitochondrial target of oxidative stress and at the same time a key contributor to it by producing reactive oxygen species. This article is part of a Special Issue entitled 'Mitochondrial function and dysfunction in neurodegeneration'.

Energy-coupling factor (ECF) transporters mediate the uptake of vitamins in bacteria. Given that these ECF transporters are not present in eukaryotic cells, they represent an interesting target for the development of novel antibiotics. Here, we present the design and synthesis of compounds that bind to ThiT, the substrate-binding domain of the ECF transporter for thiamine from Lactococcus lactis. We modified the methyl substituent of the pyrimidine ring of thiamine, in order to evaluate its contribution to the binding affinity. Our results indicate that as long as a hydrophobic substituent is maintained, the high binding affinity is almost unchanged, opening up opportunities for the design of selective compounds.

Various forms of status spongiosus occur in neonatal cattle, the best characterized of which is due to mutations of the branched-chain alpha-keto acid dehydrogenase complex (BCKD), resulting in bovine maple syrup urine disease (MSUD, branched-chain ketoaciduria). A distinctive neurological syndrome was identified between 1998 and 2003 in 9 calves in a 250-cow stabilized Gelbvieh-Red Angus herd. Both sexes were affected (6 heifers, 3 bulls), with a low annual incidence (3 cases in 1998; no cases in 1999; 2 cases in 2000; 2 in 2001; 1 in 2002; 1 in 2003). Affected calves were born full-term, unable to stand, and had constant whole-body tremors when stimulated. Animals remained in lateral recumbency until death or euthanasia; the longest survival time was 10 days postpartum. The principal histological change in 2 affected calves was diffuse, moderately severe bilaterally symmetrical status spongiosus with Alzheimer type II cells throughout the white matter of the brain. Myelin deficits were not evident and vacuoles were due to cleaved myelin sheaths. Neither recognized mutation of MSUD was identified in the E1a subunit of BCKD in 2 affected calves, 8 dams that gave birth to affected calves, a grand-dam of 3 affected calves, or a sire of 1 calf. Amino acid analysis of serum from 1 affected calf revealed normal concentrations of branched-chain amino acids, indicating that this disease is distinct from MSUD. The genetic and biochemical basis for the disorder, provisionally named congenital status spongiosus of Gelbvieh-cross cattle, is undetermined. The pattern of inheritance was not established.

Contains fulltext : 48941.pdf (Publisher’s version ) (Closed access) Cerebral developmental abnormalities occur in various inborn errors of metabolism including peroxisomal deficiencies, pyruvate dehydrogenase complex deficiency and others. Associations with abnormalities of the respiratory chain are rare. Here we report male and female siblings with microcephaly, a complex neuromigrational disorder including ependymal cysts, leptomeningeal and subcortical heterotopia, polymicrogyria, multifocal cerebral calcifications, agenesis of the corpus callosum, and spongiform changes in brainstem and cerebellum. Intractable lactic acidosis, causing death on the first day of life, was associated with severely reduced activities of complex I and complex IV. The neuropathological and biochemical findings are closely similar to those reported previously. The findings confirm a distinct genetic syndrome of disrupted brain development with TORCH-like calcifications, and a complex neuronal migration disorder associated with a multicomplex disorder of the respiratory chain.

Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI-NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo-atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low-abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI-NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.

Maple syrup urine disease is an autosomal recessive inherited disorder of branched-chain amino acid metabolism due to deficiency of the branched-chain alpha-keto acid dehydrogenase complex. The disease was originally named after the characteristic sweet aroma, reminiscent of maple syrup, present in the body fluids of affected patients. Until now, the substance responsible for the odour has not been positively identified. Using enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS), we could demonstrate that 4,5-dimethyl-3-hydroxy-2[5H]-furanone (sotolone), a well-known flavour impact compound present in fenugreek and lovage, was present in urine from seven patients with maple syrup urine disease. Urine samples from healthy control persons lacked sotolone. We have shown that sotolone is responsible for the characteristic odour of maple syrup urine disease and, since maple syrup also contains sotolone, the naming of this disease appears to be correct.

Basic regulatory properties of the 2-oxoglutarate dehydrogenase complex (OGDC) isolated and purified from the heart muscle of European bison (Bison bonasus) were studied. Kinetic studies have shown that in the absence of phosphate ions OGDC exhibits kinetic attributes of negative cooperativity with respect to 2-oxoglutarate. ADP and phosphate lower S0.5 value of OGDC for 2-oxoglutarate without changing the maximum reaction rate. NADH inhibits OGDC versus both 2-oxoglutarate and NAD+. Moreover, bison heart OGDC shows negative kinetic cooperativity for NAD+ and positive kinetic cooperativity for CoA at low CoA concentrations. The latter property has not been observed in earlier studies on OGDC from bovine and pig heart and other tissues of these animals.

We examined the effects of short-term (5 weeks) and long-term (12 weeks) physical training on actual and total activities, protein content and mRNA abundance of branched-chain 2-oxo acid dehydrogenase complex in rat skeletal muscle. The actual and total activities were significantly increased approximately 60% and approximately 40%, respectively, by long-term training. No effects of short-term training on activities were observed. The increase in the total activity corresponded to increased protein content of the E1 alpha and E2 components of the complex. On the other hand, mRNA abundance for E1 alpha and E2 were not affected by the training, but that for E1 beta was slightly, but significantly increased by both short-term and long-term trainings. These divergent alterations of the message levels for the subunits of the complex suggest that posttranslational regulatory mechanisms determine the amount of the complex in skeletal muscle. Since the complex is located in the mitochondrial matrix space, mitochondrial biogenesis in response to the training was examined by determining the content of mitochondrial DNA in the muscle. The mitochondrial DNA was proportionally increased with the total activity as well as the protein content of the complex, suggesting that expression of branched-chain 2-oxo acid dehydrogenase complex in skeletal muscle in response to physical training is associated with mitochondrial biogenesis.

Maple syrup urine disease (MSUD) is a rare disorder of branched-chain amino acids (BCAA) metabolism caused by the defective function of branched-chain α-ketoacid dehydrogenase complex (BCKD). The disease causal mutations can occur either in BCKDHA, BCKDHB or DBT genes encoding respectively the E1α, E1β and E2 subunits of the complex. In this study we report the molecular characterization of 3 Tunisian patients with the classic form of MSUD. Two novel putative mutations have been identified: the alteration c.716A>G (p.Glu239Gly) in BCKDHB and a small deletion (c.1333_1336delAATG; p.Asn445X) detected in DBT gene.

Maple Syrup Urine Disease (MSUD) is an autosomal recessive disorder caused by defects in the branched-chain α-ketoacid dehydrogenase complex resulting in accumulation of branched-chain amino acids (BCAAs) and corresponding branched-chain ketoacids (BCKAs) in tissues and plasma, which are neurotoxic. Early diagnosis and subsequent nutritional modification management can reduce the morbidity and mortality. Prior to 1990s, the diagnosis of MSUD and other inborn errors of metabolism (IEM) in Malaysia were merely based on clinical suspicion and qualitative one-dimensional thin layer chromatography technique. We have successfully established specific laboratory diagnostic techniques to diagnose MSUD and other IEM. We described here our experience in performing high-risk screening for IEM in Malaysia from 1999 to 2006. We analysed the clinical and biochemical profiles of 25 patients with MSUD.A total of 12,728 plasma and urine samples from patients suspected of having IEM were received from physicians all over Malaysia. Plasma amino acids quantitation using fully automated amino acid analyzer and identification of urinary organic acids using Gas Chromatography Mass Spectrometry (GCMS). Patients' clinical information were obtained from the request forms and case records Results: Twenty-five patients were diagnosed MSUD. Nineteen patients (76%) were affected by classical MSUD, whereas six patients had non-classical MSUD. Delayed diagnosis was common among our case series, and 80% of patients had survived with treatment with mild-to-moderate learning difficulties.Our findings suggested that MSUD is not uncommon in Malaysia especially among the Malay and early laboratory diagnosis is crucial.

Background and Methodology Pancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins. Principal Findings All tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain. Conclusions Quantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes.

Clofibrate administration to rats caused both the activation and induction of the branched-chain 2-oxo acid dehydrogenase complex in the liver; the former phenomenon occurred within the first 6 h after clofibrate administration whereas the latter occurred after 12 h. Essentially the same results were obtained with primary cultures of rat hepatocytes in the presence of 0.5 mM clofibrate, though about three-fourths of the enzyme complex in control cells (without clofibrate addition) was inactivated during a culture for 44 h, with little reduction of the enzyme amount. This was also confirmed by immunotitration analysis with antibodies raised against the purified decarboxylase and transacylase components of the enzyme complex. On the other hand, the activity of dihydrolipoamide dehydrogenase (a constituent of the complex) was little affected by clofibrate administration. The half lives of the decarboxylase and transacylase components in the primary cultures were estimated to be in the range of 22-26 h, and were unchanged in the presence of clofibrate, when determined with the use of cycloheximide and by a pulse-chase experiment. On the contrary, the rates of synthesis of these two enzyme components had increased to about 1.9-fold after 32 h cultivation in the presence of clofibrate. Thus, the increase in the synthesis of both the components resulted in induction of the complex.

Primary biliary cirrhosis (PBC) is a chronic disease characterized by portal inflammation and necrosis of small intrahepatic bile ductules (1). PBC is an irreversible condition, and destruction of the bile ductules leads to progressive cholestasis and fibrosis, and may eventually lead to the development of cirrhosis. PBC is most likely an autoimmune disorder (2), and anti-mitochondrial antibody (anti-M2) has been a diagnostically very useful marker: several studies have reported a positivity rate for anti-M2 of >95% in biopsy-confirmed PBC patients (3). The major mitochondrial antigen was identified as the 74-kDa E2 subunit of the pyruvate dehydrogenase complex (PDC), a member of the 2-oxoacid dehydrogenase complex family (4). The traditional technique for the detection of anti-M2 is immunofluorescence (5), but recently new serological assays, such as ELISA, immunoblotting, and enzyme inhibition (EI), have been developed (6)(7). A miniaturized EI assay for performance on microtiter plates has already been described (8)(9). In this study we evaluated a new commercially available and completely automated EI assay manufactured by Trace Scientific (Victoria, Australia) and compared it to the immunofluorescence and ELISA techniques performed in our laboratory. The TRACE enzymatic procedure is a unique method based on the PDC inhibitory properties of the principal anti-74-kDa antibody. PDC catalyzes the following reaction: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Pyruvate}{+}\mathrm{NAD}^{\mathrm{{+}}}{+}\mathrm{CoA}\ \begin{array}{l}\mathrm{PDC}\\{\rightarrow}\end{array}\ \mathrm{Acetyl-CoA}{+}\mathrm{NADH}{+}\mathrm{H}^{{+}}{+}\mathrm{CO}_{2}\] \end{document} In the absence of anti-M2, this reaction will proceed at the maximal rate after the addition of a serum sample. If a sample contains anti-M2, the PDC activity will be inhibited, greatly reducing the rate of reaction, typically to

Objectives An enzyme-linked immunosorbent assay (ELISA) using MESACUP-2 Test Mitochondria M2 kit (new-M2 ELISA) has recently become commercially available. The aim of this study was to evaluate the clinical utility of this newly developed ELISA for the diagnosis of primary biliary cirrhosis (PBC). Design and methods We tested the immunoreactivity of sera from 82 Japanese PBC patients to the 2-oxo-acid dehydrogenase complex (2-OADC) enzymes by indirect immunofluorescence, enzyme inhibition assay using commercially available TRACE Enzymatic Mitochondrial Antibody (M2) Assay (EMA) kit, commercial ELISAs using MESACUP Mitochondria M2 kit (old-M2 ELISA) and new-M2 ELISA, and immunoblotting on bovine heart mitochondria. Results Each test gave the following positive results; antimitochondrial antibodies (AMA) by immunofluorescence in 71 (87%) out of the 82 sera, enzymatic inhibitory antibody to pyruvate dehydrogenase complex (PDC) by EMA in 61 (74%), immunoglobulin (Ig) G class anti-PDC antibody by old-M2 ELISA in 55 (67%), IgG/M/A class anti-E2 subunit of PDC (PDC-E2)/anti-E2 subunit of branched chain oxo-acid dehydrogenase complex (BCOADC-E2)/anti-E2 subunit of 2-oxoglutarate dehydrogenase complex (OGDC-E2) antibodies by new-M2 ELISA in 73 (89%), and IgG, IgM, or IgA class antibodies against at least one of the 2-OADC enzymes by immunoblotting in 82 (100%). Fifty-three of the 82 sera (65%) were all positive by these five assays. Of the 18 sera that were positive by new-M2 ELISA but negative by old-M2 ELISA, 12 were theoretically interpretable. Of the 11 sera that were negative for AMA by immunofluorescence but positive for at least one of anti-2-OADC enzymes by immunoblotting, four (36%) were positive by new-M2 ELISA, whereas only two and one sera were positive by EMA and old-M2 ELISA, respectively. Conclusions Our results indicated that the sensitivity of the newly developed new-M2 ELISA was higher than that of EMA and old-M2 ELISA, and comparable with that of immunofluorescence. However, it is still unclear whether the new-M2 ELISA could replace the conventional immunofluorescence testing for routine assay requests because six (7%) sera showed discrepant results between these two assays.

This article has no abstract. Keywords: branched-chain α-keto acid dehydrogenase complex, human

Maple syrup urine disease (MSUD) is an autosomal recessive disorder. Impaired activity of the branched-chain 2-oxoacid dehydrogenase complex (BCOA-DH) causes accumulation of branched-chain L-amino (BCAA) and 2-oxoacids (BCOA) which may exert neurotoxic effects. Treatment comprises dietary management with strictly reduced quantities of protein and BCAA as well as aggressive intervention during acute neonatal and subsequent metabolic complications. MSUD is regarded as a metabolic disorder with potentially favourable outcome when the patients are kept on a carefully supervised long-term therapy. Up to now, three MSUD patients, exhibiting the classical form of the disease, have received orthotopic whole liver transplantation (OLT). Liver replacement resulted in a clear increase in whole body BCOA-DH activity to at least the level of very mild MSUD variants. These patients no longer require protein restricted diets and the risk of metabolic decompensation during catabolic events is apparently abolished.Considering the overall expenses, risks, and outcome, however, the benefit of OLT, even in the most severe form of MSUD, may not be significantly different from that of a classical strict dietary management. Thus, OLT appears not to represent a specific option in the treatment in MSUD.

Deficient activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC) has been documented in Alzheimer disease (AD) brain and fibroblasts. We examined glutamate metabolism in intact AD fibroblasts previously shown to have reduced KGDHC activity to determine whether this enzyme deficiency leads to a metabolic deficit in extraneural tissues. After exposure to [13N]ammonia for 5 min, the ratio of [13N]glutamate to [13N]aspartate and the ratio of unlabeled glutamate to aspartate were comparable in AD and control fibroblasts. The ratio of [13N]glutamate/[13N]glutamine was 3.7 +/- 1.8 in AD and 6.8 +/- 2.8 in control cells, but the difference was not statistically significant. Unlabeled glutamate/glutamine ratios were similar in the AD and control cells. The rate of conversion of alpha-keto[1-14C]glutarate to 14CO2 was slightly but not statistically significantly less in the AD than in the control cells. The similarity in levels of glutamate and other amino acids in intact AD and control cells may be due in part to the high glutamate and glutamine content of the culture medium used. The similar incorporation of label derived from [13N]ammonia into glutamate/aspartate and the similar levels of alpha-ketoglutarate in AD and control cells may be due in part to the fact that KGDHC is not the rate-limiting enzyme for the tricarboxylic acid cycle in fibroblasts, although it does appear to be rate-limiting in brain. Cultured cells are established tools for identifying genetic and molecular defects in the host, but the studies above show the limitations in using them as a model of brain cell metabolism.

Background: Glucose is the main source of energy for most cells. When the glucose is limited some cells can use amino acids to support their survival. This ability of oxidizing branched chain amino acids (BCAAs) of the brain metastatic breast cancer cells was investigated. Methods: Breast cancer cell lines, MDA-MB-231 cell and its brain metastatic derivative MDA-MB-Br3 were used in this study. The survivability, ability of oxidizing BCAAs and expression of BCAA oxidation related enzymes of these cells were determined. Results: MDA-MB-Br3 cells survived well and oxidized more BCAAs than MDA-MB-231 cells when cultured in glucose free medium. Accordingly, the activity of branched-chain a-ketoacid dehydrogenase complex of MDA-MB-Br3 cells is significantly higher than that of the MDA-MB-231 cells. Conclusions: The breast cancer cell line MDA-MB-Br3 can use BCAAs as a resource of energy for survival in the environment with very low level of glucose. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 984. doi:10.1158/1538-7445.AM2011-984

alpha-Ketoglutarate dehydrogenase was inactivated irreversibly and completely during oxidation of alpha-ketoadipic acid. The inactivation was revealed both in the model system with ferricyanide and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither substrate depletion nor product accumulation induced the inactivation. The results obtained were compared with recent data on the enzyme inactivation during oxidation of alpha-ketoglutaric acid. The differences in the inactivation kinetics observed with the two substrates of the enzyme were analyzed. They seem not to reflect the different mechanisms of the inactivation, but, rather, depend on the changes in the rates of the individual stages of the process.

The potential for branched-chain 2-oxo acid dehydrogenase complex (BCOADC) activity to be controlled by feedback inhibition was investigated by calculating the Elasticity Coefficients for several feedback inhibitors. We suggest that feedback inhibition is a quantitatively important regulatory mechanism by which branched-chain 2-oxo acid dehydrogenase activity is regulated. The potential for control of enzyme activity is greater for NADH than for the acyl-CoA products, and suggests that factors that alter the redox potential may physiologically regulate BCOADC activity through a feedback inhibitory mechanism in vivo. Local pH may also be an important regulatory control factor.

[2-14C]Acetate incorporation into acetyl-coenzyme A (acetyl-CoA) in wheat (Triticum aestivum L. cv Stacy) chloroplasts was inhibited by S-ethyl dipropylthiocarbamate (EPTC, 0.01 μM) and the inhibition was reversed by 90 μM acetate. [2-14C]Pyruvate was converted to acetyl-CoA by the pyruvate dehydrogenase complex (PDC) in wheat chloroplasts and the conversion was inhibited by EPTC (0.01 μM). The inhibition was reversed by 90 μM pyruvate. Production of NADH by wheat (cv. Holly) mitochondrial PDC was inhibited 10% by 3.3 10−14 M EPTC and 90% by 0.33 μM EPTC. Mitochondrial 2-oxoglutarate dehydrogenase complex activity was inhibited 65% by 0.033 μM EPTC. Dichlormid (2,2-dichloro-N,N-di-2-propenylacetamide, 0.01 μM) inhibited the incorporation of [2-14C]acetate into wheat chloroplast acetyl-CoA and the inhibition was reversed by 90 μM acetate. Dichlormid (0.01 μM) also inhibited the chloroplastic conversion of [2-14C]pyruvate into acetyl-CoA and partially reversed the inhibition by EPTC (0.1 μM) of conversion of [2-14C]pyruvate to acetyl-CoA.

A method has been developed for the activation of the branched-chain alpha-ketoacid dehydrogenase complex by alpha-chloroisocaproate, an inhibitor of branched-chain alpha-ketoacid dehydrogenase kinase in human cultured skin fibroblasts. The enzyme could be activated by pretreating the cells with alpha-chloroisocaproate before they were disrupted for measurement of the activity. After this treatment, the activity was 2- to 3-fold that of untreated cells (24.8-81.4 pmol/min per mg protein). The enzyme activity in fibroblasts from a patient with maple syrup urine disease was measured by this procedure. After activation by alpha-chloroisocaproate, the activity of fibroblasts from the patient was only 10-14 pmol/min per mg protein (10% of that of controls), and was almost the same as that of the untreated cells from this patient. These results show that it is important to consider the activation state of branched-chain alpha-ketoacid dehydrogenase complex when assaying it in disrupted cells.

Summary Hepatocytes, isolated from rats fed a low-protein diet, were incubated with [ 32 P]P i and the phosphoproteins analysed. Immunoprecipitation using antibody against El of branched-chain 2-oxo acid dehydrogenase complex demonstrated phosphorylation of the α-subunit of El. Analysis of the tryptic phosphopeptides from the α-subunit indicated that two sites were phosphorylated. 4-methyl 2-oxopentanoate and DL-2-chloro 4-methylpentanoate decreased labelling of both sites. No major direct effects of several hormones on phosphorylation of branched-chain 2-oxo acid dehydrogenase was observed.

In rats fed a high-protein diet, the branched-chain 2-oxo-acid dehydrogenase complex in liver was essentially fully acitve and its activity state was unaffected by subsequent starvation for 48 h. Feeding with a low-protein diet led to a decrease in the activity state which was essentially reversed by 48 h of starvation. In heart, the enzyme was primarily inactive (activity state 18%) in rats fed a high-protein diet, with both low-protein diet and starvation leading to a further decrease in the activity state.

After electrophoresis impure transketolase preparations stained readily with an activity stain based on a published method, but pure preparations gave no reaction. The bands first obtained were due to the presence of (i) a transketolase- d -glyceraldehyde-3-phosphate dehydrogenase complex, (ii) alcohol dehydrogenases, and (iii) a zone of high local pH at the buffer front. Lyophilization of the reagents eliminated artifacts due to alcohol dehydrogenase. An external starch indicator gel was developed that gave colored bands with both pure and impure transketolase preparations and a similar indicator gel was developed for transaldolase.

A complex between partially purified d(-)-β-hydroxybutyrate dehydrogenase from beef heart mitochondria and soybean lecithin was purified. The active complex contains a single type of polypeptide chain of molecular weight 32,000 as determined by sodium dodecyl sulfate-acrylamide gel electrophoresis. The amino acid composition of the polypeptide chain is similar to that of many soluble enzymes, despite the very strong interaction with lecithin vesicles which was demonstrated. Cross-linking experiments with dimethyl suberimidate dihydrochloride failed to disclose any polypeptide aggregates in the enzyme-lecithin complex. The apparent Michaelis constants for all four substrates and the temperature dependence of the initial velocity were determined for the purified complex.