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1.
Funchal C Gottfried C de Almeida LM dos Santos AQ Wajner M Pessoa-Pureur R 《Cellular and molecular neurobiology》2005,25(5):851-867
1. Maple syrup urine disease (MSUD) is an inherited metabolic disorder predominantly characterized by neurological dysfunction and cerebral atrophy whose patophysiology is poorly known. 2. We investigated here whether the branched-chain amino acids (BCAA) leucine (Leu), isoleucine (Ile) and valine (Val), which are the biochemical hallmark of this disorder, could alter astrocyte morphology and cytoskeleton reorganization by exposing cultured astrocytes from cerebral cortex of neonatal rats to various concentrations of the amino acids. A change of cell morphology from the usual polygonal to the appearance of fusiform or process-bearing cells was caused by the BCAA. Cell death was also observed when astrocytes were incubated in the presence of BCAA for longer periods. 3. Val-treated astrocytes presented the most dramatic morphological alterations. Immunocytochemistry with anti-actin and anti-GFAP antibodies revealed that all BCAA induced reorganization of actin and GFAP cytoskeleton. In addition, lysophosphatidic acid, an activator of RhoA GTPase pathway, was able to totally prevent the morphological alterations and cytoskeletal reorganization induced by Val, indicating that the RhoA signaling pathway was involved in these effects. 4. Furthermore, creatine attenuated the morphological alterations provoked by the BCAA, the protection being more pronounced for Val, suggesting that impairment of energy homeostasis is partially involved in BCAA cytotoxic action. The data indicate that the BCAA accumulating in MSUD are toxic to astrocyte cells, a fact that may be related to the pathogenesis of the neurological dysfunction of MSUD patients. 相似文献
2.
Schuck PF Tonin A da Costa Ferreira G Viegas CM Latini A Duval Wannmacher CM de Souza Wyse AT Dutra-Filho CS Wajner M 《Cellular and molecular neurobiology》2007,27(1):147-160
Growing evidence indicates that some metabolites derived from the kynurenine pathway, the major route of l-tryptophan catabolism, are involved in the neurotoxicity associated with several brain disorders, such as Huntington’s disease,
Parkinson’s disease and Alzheimer’s disease, as well as in glutaryl-CoA dehydrogenase deficiency (GAI). Considering that the
pathophysiology of the brain damage in these neurodegenerative disorders is not completely defined, in the present study,
we investigated the in vitro effect of l-kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HA) and anthranilic acid (AA)
on some parameters of energy metabolism, namely glucose uptake, 14CO2 production from [U-14C] glucose, [1-14C] acetate and [1,5-14C] citrate, as well as on the activities of the respiratory chain complexes I–IV and Na+,K+-ATPase activity in cerebral cortex from 30-day-old rats. We observed that all compounds tested, except l-kynurenine, significantly increased glucose uptake and inhibited 14CO2 production from [U-14C] glucose, [1-14C] acetate and [1,5-14C] citrate. In addition, the activities of complexes I, II and IV of the respiratory chain were significantly inhibited by
3HK, while 3HA inhibited complexes I and II activities and AA inhibited complexes I–III activities. Moreover, Na+,K+-ATPase activity was not modified by these kynurenines. Taken together, our present data provide evidence that various kynurenine
intermediates provoke impairment of brain energy metabolism. 相似文献
3.
Kawana S Nakagawa K Hasegawa Y Yamaguchi S 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(30):3113-3118
A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using 2H8-Val, 2H3-Leu, 2H3-Met and 2H5-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients = 1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood. 相似文献
4.
Sgaravatti AM Rosa RB Schuck PF Ribeiro CA Wannmacher CM Wyse AT Dutra-Filho CS Wajner M 《Biochimica et biophysica acta》2003,1639(3):232-238
Neurological dysfunction is a common finding in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly known. In the present study, we investigated the effect of the in vitro effect of the branched chain alpha-keto acids (BCKA) accumulating in MSUD on some parameters of energy metabolism in cerebral cortex of rats. [14CO(2)] production from [14C] acetate, glucose uptake and lactate release from glucose were evaluated by incubating cortical prisms from 30-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of 1-5 mM of alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV) or alpha-ketoisovaleric acid (KIV). All keto acids significantly reduced 14CO(2) production by around 40%, in contrast to lactate release and glucose utilization, which were significantly increased by the metabolites by around 42% in cortical prisms. Furthermore, the activity of the respiratory chain complex I-III was significantly inhibited by 60%, whereas the other activities of the electron transport chain, namely complexes II, II-III, III and IV, as well as succinate dehydrogenase were not affected by the keto acids. The results indicate that the major metabolites accumulating in MSUD compromise brain energy metabolism by blocking the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients. 相似文献
5.
The aim of this study was to examine whether flux through the pathways of carbohydrate oxidation is accurately reflected in the pattern of 14CO2 release from positionally labelled [14C]substrates in conventional radiolabel feeding studies. Heterotrophic cell suspension cultures of Arabidopsis thaliana were used for this work. The presence of an alkaline trap to capture metabolically generated 14CO2 had no significant effect on the ratio of 14CO2 release from specifically labelled [14C]substrates, or on the metabolism of [U-14C]glucose by the cells. Although the amount of 14CO2 captured in a conventional time-course study was only about half of that released from a sample acidified at an equivalent time point, the ratios of 14CO2 released from different positionally labelled [14C]glucose and [1-14C]gluconate were the same in untreated and acidified samples. Less than 5% of radioactivity supplied to the growth medium as [14C]bicarbonate was incorporated into acid-stable compounds, and there was no evidence for appreciable reassimilation of 14CO2 generated intracellularly during oxidation of [1-14C]gluconate by the cells. It is concluded that the ratio of label captured from specifically labelled [14C]glucose is a valid and convenient measure of the relative rates of oxidation of the different positional carbon atoms within the supplied respiratory substrate. However, it is argued that failure to compensate for the incomplete absorption of 14CO2 by an alkaline trap may distort estimates of respiration that rely on an absolute measure of the amount of 14CO2 generated by metabolism. 相似文献
6.
《Plant science》1986,45(1):31-36
More than 80% of the radioactivity from [U-14C]glucose metabolised by anaerobic rice seedlings or by excised roots or coleoptiles was recovered as ethanol plus CO2; less than 5% was recovered as water-soluble acidic components. Rates of 14CO2 formation from [U-14C]glucose were similar in roots and coleoptiles in both N2 and air atmospheres. More 14CO2 was formed from [U-14C]glucose than could be accounted for by ethanolic fermentation, and the specific yields of 14CO2 from [6-14C]glucose and [1-14C]glucose gave unusually high C-6/C-1 ratios (1.7) in the anaerobic coleoptile. The results may indicate that appreciable pentan synthesis occurs in the anaerobic coleoptile. 相似文献
7.
Gloaguen M Le Floc'h N Brossard L Barea R Primot Y Corrent E van Milgen J 《Animal : an international journal of animal bioscience》2011,5(11):1734-1742
The branched-chain amino acids (BCAA) valine (Val) and isoleucine (Ile) are considered to be among the next-limiting amino acids for growth in piglets. In earlier studies, we estimated the standardized ileal digestible (SID) Val : Lys (lysine) requirement to be at least 70%, whereas the Ile : Lys requirement may be as low as 50%. Because the BCAA partially share a common route of catabolism, the supply of one BCAA may affect the availability of the other BCAA. Four experiments were conducted to determine the response of 6-week-old piglets to the Val supply in relation to the other BCAA. A deficient supply of Val or Ile typically results in a reduction in average daily feed intake (ADFI). Experiment 1 was designed to determine the effect of a limiting Val supply, independent of the effect on feed intake. In a dose-response study using restrictively fed piglets, nitrogen retention did not increase for an SID Val : Lys supply greater than 64%. In the remaining experiments, piglets were offered feed ad libitum using ADFI, average daily gain (ADG) and gain-to-feed ratio as response criteria. The interaction between the Val and leucine (Leu) was studied in Experiment 2 in a 2 × 2 factorial design (60% and 70% SID Val : Lys, and 111% and 165% SID Leu : Lys). Performance was considerably lower in piglets receiving 60% Val : Lys compared with those receiving 70% Val : Lys and was lowest in piglets receiving the diet with low Val and high Leu content. To further evaluate the interaction between Val and Leu, a dose-response study was carried out in which the response to Val supply was studied in combination with high Leu supply (165% Leu : Lys). Using a curvilinear-plateau model, the average SID Val : Lys requirement was 72%. However, low Val supply (60% SID Val : Lys) reduced performance by 13% to 38%, which was much greater than what we observed in earlier studies. Experiment 4 was carried out to test the hypothesis that the Val requirement is not affected by low Ile supply (50% SID Ile : Lys). Performance was not improved for Val : Lys supplies greater than 65%, which may indicate that Ile (and not Lys) was second-limiting in this study. In conclusion, the first response of piglets to deficient Val supply appears to be a reduction in ADFI, rather than a reduction in ADG or nitrogen retention. A large supply of Leu may not affect the Val requirement per se, but may aggravate the consequences of Val deficiency. 相似文献
8.
Claude D. Stringer I.Lucile Norton Fred C. Hartman 《Archives of biochemistry and biophysics》1981,208(2):495-501
Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [3H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys1Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys1AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys1 ThrThrAsxAsxPheThrArg AlaCys1ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys1HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase. 相似文献
9.
Acetylcholine Synthesis and CO2 Production from Variously Labeled Glucose in Rat Brain Slices and Synaptosomes 总被引:4,自引:4,他引:0
Abstract: The molecular basis of the close linkage between oxidative metabolism and acetylcholine (ACh) synthesis is still unclear. We studied this problem in slices and synaptosomes by measurement of ACh synthesis from [U-14C]glucose, and 14CO2 production from [3,4-14C]- and [2-14C]glucose, an index of glucose decarboxylation by the pyruvate dehydrogenase complex (PDH) and the enzymes of the Krebs cycle, respectively. We examined both under conditions that either inhibited (low O2 or antimycin) or stimulated (2,4- dinitrophenol [DNP] or 35 mm -K+) 14CO2 production from [2-14C]- or [3,4-14C]glucose. Incorporation of [U-14C]glucose into ACh was reduced under low O2 and by antimycin or DNP (by 51-93%) and stimulated by 35 mm -K+ (by 30-60%). Under all of these conditions, ACh synthesis and the decarboxylation of [3,4-14C]- and [2-14C]glucose were linearly related (r= 0.741 and 0.579, respectively). The difference in the rate of 14CO2 production from [3,4-14C]- and [2-14C]glucose was used as a measure of the amount of glucose that was not oxidatively decarboxylated (efflux). We found that efflux was reduced (low 02 and antimycin), unchanged (DNP in slices), or increased (DNP in synaptosomes and K+ stimulation in slices) compared with control values under 100% O2. ACh synthesis and efflux were more closely related (r= 0.860) than ACh synthesis and 14CO2 production from variously labeled glucoses. 相似文献
10.
Energy metabolism in hypoxic astrocytes: Protective mechanism of fructose-1,6-bisphosphate 总被引:2,自引:0,他引:2
Judith A. Kelleher Pak H. Chan Thelma Y. Y. Chan George A. Gregory 《Neurochemical research》1995,20(7):785-792
The protective effects of fructose-1,6-biphosphate (FBP) during hypoxia/ischemia are thought to result from uptake and utilization of FBP as a substrate for glycolysis or from stimulation of glucose metabolism. To test these hypotheses, we measumed CO2 and lactate production from [6-14C]glucose, [1-14C]glucose, and [U-14C]FBP in normoxic and hypoxic cultured astrocytes with and without FBP present. FBP had little effect on CO2 production by glycolysis, but increased CO2 production by the pentose phosphate pathway. Labeled FBP produced very small amounts of CO2. Lactate production from [1-, and 6-14C]glucose increased similarly during hypoxic hypoxia; the increase was independent of added FBP. Labeled lactate from [U-14C]FBP was minimal. We conclude that exogenous FBP is not used by astrocytes as a substrate for glycolysis and that FBP alters glucose metabolism. 相似文献
11.
Yohei Miyanoiri Mitsuhiro Takeda Kosuke Okuma Akira M. Ono Tsutomu Terauchi Masatsune Kainosho 《Journal of biomolecular NMR》2013,57(3):237-249
The 1H–13C HMQC signals of the 13CH3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ1-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically 13CH3-labeled [U–2H;15N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G. 相似文献
12.
Abstract— Replacement of bicarbonate-Locke incubation medium with feline CSF reduced [14C]ACh formation from [U-14C]glucose by rat brain mince approx 30%. CSF was obtained from a cannula leading to the cisterna magna of freely moving cats. The component of CSF responsible for inhibition was characterized as a dialyzable heat-stable organic anion. Choline acetyltransferase activity was not altered by CSF. [14C]ACh synthesis and 14CO2 production from [U-14C]glucose but not from [2-14C]-pyruvate were inhibited by CSF, suggesting inhibition in the metabolism of glucose to pyruvate. The anionic fraction of human CSF was as potent as that from feline CSF in inhibiting 14CO2 production from [U-14C]glucose. Brain hexokinase was inhibited by the anionic fraction of feline CSF. The inhibition was non-competitive with respect to glucose and uncompetitive with respect to ATP. It is suggested that inhibition of hexokinase by CSF was responsible at least in part for the inhibition of glucose metabolism which resulted in decreased [14C]ACh synthesis and 14CO2 production. 相似文献
13.
Thureen PJ Anderson SM Hay WW 《American journal of physiology. Regulatory, integrative and comparative physiology》2000,279(3):R849-R859
We tested the hypothesis that decreased fetal amino acid (AA) supply, produced by maternal hypoaminoacidemia (low AA) during hyperglycemia (HG), is reversible with maternal AA infusion and regulates fetal insulin concentration ([I]). We measured net uterine and umbilical AA uptakes during maternal HG/low AA concentration ([AA]) and after maternal intravenous infusion of a mixed AA solution. After 5 days HG, all maternal [AA] except glycine were decreased >50%, particularly essential [AA] (P < 0.00005). Most fetal [AA] also were decreased, especially branched-chain AA (P < 0.001). Maternal AA infusion increased net uterine uptakes of Val, Leu, Ile, Met, and Ser and net umbilical uptakes of Val, Leu, Ile, Met, Phe, and Arg but did not change net uteroplacental uptake of any AA. Fetal [I] increased 55 +/- 14%, P < 0.001, with correction of fetal [AA], despite the lack of change in fetal glucose concentration. Thus generalized maternal hypoaminoacidemia decreases uterine and umbilical uptakes of primarily the essential AA and decreases fetal branched-chain [AA]. These changes are reversed with correction of maternal [AA], which also increases fetal [I]. 相似文献
14.
The epimastigote or culture form of Trypanosoma cruzi oxidizes [3-14C] pyruvate and [2-14C] acetate to 14CO2 without an apparent increase in overall respiration. This oxidation takes place through the tricarboxylic acid cycle as shown by (a) the incorporation of substrate 14C into cycle intermediates; (b) the earlier liberation of acetate carboxyl carbon as CO2; and (c) the characteristic intramolecular distribution of pyruvate and acetate carbon atoms in the skeletal carbon of aspartic and glutamic acids. Upon oxidation of [3-14C] pyruvate and [2-14C] acetate, two of the products, alanine and glutamic acid, are found to account for more than 50% of incorporated 14C; labeling of alanine predominates with [3-14C] pyruvate while labeling of glutamic acid predominates with [2-14C] acetate. Using [1- or 6-14C] glucose as substrate, the pattern of 14C distribution in soluble metabolites closely resembles that obtained with [3-14C] pyruvate, in accordance with the joint operation of the Embden-Meyerhof pathway and Krebs cycle. The cycle operation depends on electron transport through the mitochondrial respiratory chain, since antimycin A, at a relatively low concentration, inhibits the oxidation of [2-14C] acetate to 14CO2, to the same extent as the parasite respiration. Though functional in T. cruzi epimastigotes, the oxidative role of the Krebs’ cycle is apparently limited by the absence of an efficient oxidative apparatus. The cycle operation does, however, constitute an important source of skeletal carbon for the biosynthesis of amino acids and can contribute to the process of glycogenesis. 相似文献
15.
Brain cortex slices from fed, 48 h and 120 h fasted rats were incubated and 14CO2 was measured from (a) [U-14C]glucose (5 mm ) either alone or in the presence of l -lcucine (0.1 or 1 mm ), and (b) [U-14C]leucine or [l-14C]leucine at 0.1 or 1 mm with or without glucose (5 mm ). In other experiments, sodium dl -3-hydroxybutyrate (3-OHB) or acetoacetate (AcAc) at 1 or 5 mm were added in the above incubation mixture. The rate of conversion of [U14C]glucose to CO2 was decreased 20% by leucine at 1 mm and 30–50% by 3-OHB at 1 or 5 mm but not by leucine at 0.1 mm . The effects of 3-OHB and of leucine (1 mm ) were not additive. The effects of leucine were similar in the fed and fasted rats. The rate of conversion of [U-14C]leucine or [l-,4C]leucine to 14CO2 at 0.1 mm and 1.0 mm was increased by glucose (35%) in the fed or fasted rats. Ketone bodies in the absence of glucose had no effect on leucine oxidation. However, the stimulatory effect of glucose on the rate of conversion of leucine to CO2 was inhibited by 3-OHB at 5 mm . These results suggest that (a) leucine in increased concentrations (1 mm ) may reduce glucose oxidation by brain cortex while itself becoming an oxidative fuel for brain, and (b) leucine oxidation by brain may be influenced by the prevailing glucose and ketone concentrations. 相似文献
16.
Lactobacillus plantarum was grown in complex media containing glucose and yeast extract. The maximum growth yield based on yeast extract consumption was 0.5 g dwt g-1. Growth yield YATP 15–17 g dwt mol ATP-1 was almost constant in the glucose limited A-stat experiment whereas in the yeast extract limited culture it increased with dilution rate. The maximum specific growth rate observed, 0.5 h-1, was similar for both A-stat and batch cultures. Specific oxygen consumption, QO2, reached the value of 1.8 mmol O2 h-1 g dwt-1. It was shown that Val, Ile, Leu, Tyr and Phe, were consumed mainly as free amino acids, while Asp, Pro, Lys and Arg were derived from peptides. Significantly more Asp, Ser, Glu, Val, Ile, Leu and Phe were consumed than needed to build up cell protein whereas some Pro, Gly, Ala and Lys was synthesized. A network of metabolic reactions in L. plantarum was proposed on the basis of the experimental data. 相似文献
17.
T. N. DARLING D. G. DAVIS R. E. LONDON J. J. BLUM 《The Journal of eukaryotic microbiology》1989,36(2):217-225
13C-nuciear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of 14CO2 production from 14C-labeIed substrate. Cells incubated with [2-13C]glycerol released acetate, succinate and D-lactate in addition to CO2. Cells incubated with acetate released only CO2. More succinate C-2/C-3 than C-l/C-4 was released from both [2-13C]glycerol and [2-13C]glucose, indicating that succinate was formed predominantly by CO2 fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of 14CO2 production from [l,(3)-14C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased 14CO2 production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of 14CO2 production from [U-14C]- and [l-14C]alanine by about 20%. This is consistent with rapid equilibration of alanine with pyruvate derived from glucose and yet little decrease in the specific activity of the large alanine pool. 相似文献
18.
Lixiang Chen Peng Li Junjun Wang Xilong Li Haijun Gao Yulong Yin Yongqing Hou Guoyao Wu 《Amino acids》2009,37(1):143-152
This study was conducted using the piglet model to test the hypothesis that mucosal cells of the neonatal small intestine
can degrade nutritionally essential amino acids (EAA). Enterocytes were isolated from the jejunum of 0-, 7-, 14-, and 21-day-old
pigs, and incubated for 45 min in Krebs buffer containing plasma concentrations of amino acids and one of the following L-[1-14C]- or L-[U-14C]-amino acids plus unlabeled tracees at 0.5, 2, or 5 mM: histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan and valine. In these cells, branched-chain amino acids (BCAA) were extensively transaminated and 15–50%
of decarboxylated branched-chain α-ketoacids (BCKA) were oxidized to CO2 depending on the age of piglets. BCAA transamination increased but their decarboxylation decreased between 0 and 14 days
of age. Addition of 1 and 2 mM α-ketoglutarate to incubation medium dose-dependently stimulated BCAA transamination without
affecting their decarboxylation. Western blot analysis revealed that the abundance of mitochondrial BCAA aminotransferase
declined but cytosolic BCAA aminotransferase increased between 0 and 14 days of age, with the cytosolic protein being the
major isoform in 7- to 21-day-old pigs. BCKA dehydrogenase protein existed primarily as the phosphorylated (inactive) form
in enterocytes of newborn pigs and its levels were markedly reduced in older pigs. All measured parameters of BCAA metabolism
did not differ between 14- and 21-day-old pigs. In contrast to BCAA, catabolism of methionine and phenylalanine was negligible
and that of other EAA was absent in enterocytes from all ages of piglets due to the lack of key enzymes. These results indicate
that enterocytes are an important site for substantial degradation of BCAA but not other EAA in the neonatal gut. 相似文献
19.
NH2-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH2-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH2-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala2Val3 residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH2-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH2-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH2-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site. 相似文献
20.
Respiration studies in vitro, in which tissue slices were incubated with [1-14C]glucose or [6-14C]glucose and 14CO2 collected, resulted in C-1/C-6 14CO2 ratios that were higher in slices of tumor and newborn brain than in slices of adult brain. In adult brain, the C-1/C-6 14CO2 ratio averaged close to unity. In slices of tumor and newborn brain however, the mean C-1/C-6 ratio was greater than three. Addition of phenazine methosulfate (PMS) increased conversion of [1-14C]glucose to 14CO2 in slices of normal adult brain 5-fold, and in slices of newborn brain and tumor, approx 12-fold. Injection of animals with 6-aminonicotinamide (6-AN) decreased conversion of [1-14C]glucose in slices of normal brain 30% but decreased conversion in tumor slices by 80%. Evidence supports the presence of an active hexose monophosphate pathway (HMP) in tumors of the nervous system regulated in part by available NADP+ levels. Inhibition by 6-AN was more effective in tumors than in normal adult brain. 相似文献