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Journal of Animal Science Aug 2013The objective of this study was to determine the Ile requirement in early (d 39 to 61) and late (d 89 to 109) pregnancy using the indicator AA oxidation method. The same... (Randomized Controlled Trial)
Randomized Controlled Trial
The objective of this study was to determine the Ile requirement in early (d 39 to 61) and late (d 89 to 109) pregnancy using the indicator AA oxidation method. The same 7 Large White × Landrace sows in their fourth parity were used in early and late pregnancy. Each sow received 6 diets based on corn, corn starch, and sugar in both early and late pregnancy at constant feed allowances (2.5 kg/d). Diets provided Ile at 20, 40, 60, 80, 100, and 120% of the Ile requirement (6.2 g/d based on the 1998 NRC) in early and 60, 80, 100, 140, 160, and 180% in late pregnancy. After determination of (13)C background in expired CO2 and plasma free Phe for 1.5 h when confined in respiration chambers, sows were fed the tracer, L[1-(13)C]Phe, a rate of 2.0 mg/(kg BW·h) over 4 h divided into eight 30-min meals. Expired CO2 and plasma free Phe were analyzed for (13)C enrichment above background. Requirements were determined as the breakpoint in 2-phase nonlinear models. Sow BW was 246.5 kg in early and 271.6 kg in late pregnancy. Daily gain of the 6 sows was similar in early (344 g/d) and late pregnancy (543 g/d). During pregnancy, sow maternal gain was 19.1 ± 4.4 kg and litters of 17.7 ± 0.8 piglets weighed 22.6 ± 0.9 kg at birth. The Ile requirement was 3.6 ± 1.2 g/d (P = 0.001) in early pregnancy with a Phe retention (-0.59 g/d) and energy retention (-0.31 MJ/d) that were not different from 0. This indicates that the fourth parity sows had requirements close to maintenance in early pregnancy. The Ile requirement in late pregnancy was 9.7 ± 1.9 g/d (P = 0.001) when sows retained 3.30 g/d of Phe and -1.45 MJ/d of energy. The greater Ile requirement in late pregnancy was probably caused by the increased conceptus growth after d 70 of pregnancy. Phenylalanine flux, oxidation, and nonoxidative disposal increased (P < 0.1) from early to late pregnancy, but body protein breakdown did not. Phenylalanine oxidation, nonoxidative disposal, and retention increased (P < 0.01) with increasing Ile intake in early pregnancy but were not affected by Ile intake in late pregnancy. Body protein breakdown did not respond to Ile intake in early or late pregnancy. Although energy retention was similar in early and late pregnancy, the respiratory quotient decreased (P = 0.047) from early (1.05) to late pregnancy (0.98), indicating lipid mobilization in late pregnancy when Ile was at or above the requirement. The results of this study show that the Ile requirement of sows increases from early to late pregnancy.
Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Diet; Female; Isoleucine; Nutritional Requirements; Pregnancy; Pregnancy, Animal; Swine
PubMed: 23658325
DOI: 10.2527/jas.2012-6174 -
Journal of Bacteriology Mar 1969Microorganisms in ruminal ingesta and pure cultures of anaerobic ruminal bacteria of different physiological and morphological groups incorporated (14)C from labeled...
Microorganisms in ruminal ingesta and pure cultures of anaerobic ruminal bacteria of different physiological and morphological groups incorporated (14)C from labeled 2-methylbutyrate during growth. The radioactivity was incorporated mainly into lipid and protein. Isoleucine was the only labeled amino acid found in acid hydrolysates of protein from either pure or mixed cultures. Radioactivity in isoleucine synthesized from 2-methylbutyrate-1-(14)C was entirely in carbon-2. Thus, the carboxylation of 2-methylbutyrate is a pathway for synthesis of isoleucine different from that operative in many aerobic and facultative microorganisms. The specific activity of isoleucine from 2-methylbutyrate by Bacteroides rumminicola 23 increased with higher concentrations of 2-methylbutyrate (2.6 to 44 x 10(-5)m) in the growth medium. At the highest concentration, the specific activity of isoleucine synthesized was 40% of the specific activity of the 2-methylbutyrate in the growth medium. The use of enzymatic casein hydrolysate, oxytocin, or vasopressin rather than ammonia as nitrogen source for growth of strain 23 depressed the incorporation of 2-methylbutyrate into isoleucine. Synthesis of isoleucine from 2-methylbutyrate appears to be an important reaction in the rumen.
Topics: Animals; Bacteria; Bacterial Proteins; Bacteroides; Butyrates; Carbon Isotopes; Isoleucine; Lipid Metabolism; Peptides; Peptostreptococcus; Rumen
PubMed: 5813342
DOI: 10.1128/jb.97.3.1220-1226.1969 -
Microbiology (Reading, England) Feb 2010Cyanothece sp. ATCC 51142 is an aerobic N(2)-fixing and hydrogen-producing cyanobacterium. Isotopomer analysis of its amino acids revealed an identical labelling profile...
Cyanothece sp. ATCC 51142 is an aerobic N(2)-fixing and hydrogen-producing cyanobacterium. Isotopomer analysis of its amino acids revealed an identical labelling profile for leucine and isoleucine when Cyanothece 51142 was grown mixotrophically using 2-(13)C-labelled glycerol as the main carbon source. This indicated that Cyanothece 51142 employs the atypical alternative citramalate pathway for isoleucine synthesis, with pyruvate and acetyl-CoA as precursors. Utilization of the citramalate pathway was confirmed by an enzyme assay and LC-MS/MS analysis. Furthermore, the genome sequence of Cyanothece 51142 shows that the gene encoding the key enzyme (threonine ammonia-lyase) in the normal isoleucine pathway is missing. Instead, the cce_0248 gene in Cyanothece 51142 exhibits 53 % identity to the gene encoding citramalate synthase (CimA, GSU1798) from Geobacter sulfurreducens. Reverse-transcription PCR indicated that the cce_0248 gene is expressed and its transcriptional level is lower in medium with isoleucine than in isoleucine-free medium. Additionally, a blast search for citramalate synthase and threonine ammonia-lyase implies that this alternative isoleucine synthesis pathway may be present in other cyanobacteria, such as Cyanothece and Synechococcus. This suggests that the pathway is more widespread than originally thought, as previous identifications of the citramalate pathway are limited to mostly anaerobic bacteria or archaea. Furthermore, this discovery opens the possibility that such autrotrophic micro-organisms may be engineered for robust butanol and propanol production from 2-ketobutyrate, which is an intermediate in the isoleucine biosynthesis pathway.
Topics: Bacterial Proteins; Carbon Isotopes; Cyanobacteria; Isoleucine; Malates
PubMed: 19875435
DOI: 10.1099/mic.0.031799-0 -
Biotechnology and Applied Biochemistry Jan 2019Cysteine synthase A (CysK) catalyzes the last reaction of l-cysteine synthesis in bacteria, but its moonlighting functions have been revealed recently. In this study,...
Cysteine synthase A (CysK) catalyzes the last reaction of l-cysteine synthesis in bacteria, but its moonlighting functions have been revealed recently. In this study, CysK was overexpressed in Corynebacterium glutamicum IWJ001, an l-isoleucine producer. Compared with the control IWJ001/pDXW-8, IWJ001/pDXW-8-cysK cells grew fast during log phase, and produced 26.5% more l-isoleucine in flask fermentation and 23.5% more l-isoleucine in fed-batch fermentation. The key genes aspC, lysC, hom, thrB, ilvA, and ilvBN involved in l-isoleucine biosynthesis were all upregulated in IWJ001/pDXW-8-cysK, compared with IWJ001/pDXW-8. In addition, IWJ001/pDXW-8-cysK cells were longer and thicker than IWJ001/pDXW-8 cells. Compared with IWJ001/pDXW-8, the membrane permeability increased 15.8% and biofilm formation ability decreased 71.3% for IWJ001/pDXW-8-cysK cells. The results demonstrate that CysK overexpression in C. glutamicum is a good approach to enhance l-isoleucine production.
Topics: Bacterial Proteins; Corynebacterium glutamicum; Cysteine Synthase; Gene Expression; Isoleucine
PubMed: 30311712
DOI: 10.1002/bab.1698 -
Diabetes Research and Clinical Practice Jan 2021In healthy individuals, intragastric administration of the branched-chain amino acids, leucine and isoleucine, diminishes the glycaemic response to a mixed-nutrient... (Randomized Controlled Trial)
Randomized Controlled Trial
Intragastric administration of leucine and isoleucine does not reduce the glycaemic response to, or slow gastric emptying of, a carbohydrate-containing drink in type 2 diabetes.
AIMS
In healthy individuals, intragastric administration of the branched-chain amino acids, leucine and isoleucine, diminishes the glycaemic response to a mixed-nutrient drink, apparently by stimulating insulin and slowing gastric emptying, respectively. This study aimed to evaluate the effects of leucine and isoleucine on postprandial glycaemia and gastric emptying in type-2 diabetes mellitus (T2D).
METHODS
14 males with T2D received, on 3 separate occasions, in double-blind, randomised fashion, either 10 g leucine, 10 g isoleucine or control, intragastrically 30 min before a mixed-nutrient drink (500 kcal; 74 g carbohydrates, 18 g protein, 15 g fat). Plasma glucose, insulin and glucagon were measured from 30 min pre- until 120 min post-drink. Gastric emptying of the drink was also measured.
RESULTS
Leucine and isoleucine stimulated insulin, both before and after the drink (all P < 0.05; peak (mU/L): control: 70 ± 15; leucine: 88 ± 17; isoleucine: 74 ± 15). Isoleucine stimulated (P < 0.05), and leucine tended to stimulate (P = 0.078), glucagon before the drink, and isoleucine stimulated glucagon post-drink (P = 0.031; peak (pg/mL): control: 62 ± 5; leucine: 70 ± 9; isoleucine: 69 ± 6). Neither amino acid affected gastric emptying or plasma glucose (peak (mmol/L): control: 12.0 ± 0.5; leucine: 12.5 ± 0.7; isoleucine: 12.0 ± 0.6).
CONCLUSIONS
In contrast to health, in T2D, leucine and isoleucine, administered intragastrically in a dose of 10 g, do not lower the glycaemic response to a mixed-nutrient drink. This finding argues against a role for 'preloads' of either leucine or isoleucine in the management of T2D.
Topics: Adult; Aged; Amino Acids, Branched-Chain; Blood Glucose; Cross-Over Studies; Diabetes Mellitus, Type 2; Dietary Supplements; Double-Blind Method; Energy Drinks; Gastric Emptying; Humans; Isoleucine; Leucine; Male; Middle Aged; Postprandial Period
PubMed: 33310174
DOI: 10.1016/j.diabres.2020.108618 -
Journal of Bacteriology Sep 1971Isoleucine hydroxamate (Ile.Hdx) was found to inhibit the growth of Serratia marcescens and to antagonize isoleucine. At a low concentration of Ile.Hdx, at which the...
Isoleucine hydroxamate (Ile.Hdx) was found to inhibit the growth of Serratia marcescens and to antagonize isoleucine. At a low concentration of Ile.Hdx, at which the growth of the wild strain was completely inhibited, the growth of an isoleucine auxotroph was not inhibited in the medium containing a limiting amount of d-threonine as the isoleucine source. At a higher concentration, this antagonist exhibited a considerable inhibitory effect on the growth of the auxotroph. Ile.Hdx showed the same inhibitory effect as isoleucine on l-threonine dehydratase activity at the concentrations 10 times those of isoleucine. Ile.Hdx caused also derepression of isoleucine-valine biosynthetic enzymes and the derepression was overcome by isoleucine. These results indicate the Ile.Hdx causes growth inhibition by its effects on isoleucine metabolism.
Topics: Chemical Phenomena; Chemistry; Culture Media; Densitometry; Hydro-Lyases; Hydroxamic Acids; Isoleucine; Ligases; Serratia marcescens; Threonine; Transaminases; Valine
PubMed: 4937785
DOI: 10.1128/jb.107.3.741-745.1971 -
Aging Cell Jun 2022The proportion of humans suffering from age-related diseases is increasing around the world, and creative solutions are needed to promote healthy longevity. Recent work... (Review)
Review
The proportion of humans suffering from age-related diseases is increasing around the world, and creative solutions are needed to promote healthy longevity. Recent work has clearly shown that a calorie is not just a calorie-and that low protein diets are associated with reduced mortality in humans and promote metabolic health and extended lifespan in rodents. Many of the benefits of protein restriction on metabolism and aging are the result of decreased consumption of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we discuss the emerging evidence that BCAAs are critical modulators of healthy metabolism and longevity in rodents and humans, as well as the physiological and molecular mechanisms that may drive the benefits of BCAA restriction. Our results illustrate that protein quality-the specific composition of dietary protein-may be a previously unappreciated driver of metabolic dysfunction and that reducing dietary BCAAs may be a promising new approach to delay and prevent diseases of aging.
Topics: Amino Acids, Branched-Chain; Diet, Protein-Restricted; Isoleucine; Leucine
PubMed: 35526271
DOI: 10.1111/acel.13626 -
Journal of the American Chemical Society Jan 2016The nonproteinogenic amino acid L-allo-isoleucine (L-allo-Ile) is featured in an assortment of life forms comprised of, but not limited to, bacteria, fungi, plants and...
The nonproteinogenic amino acid L-allo-isoleucine (L-allo-Ile) is featured in an assortment of life forms comprised of, but not limited to, bacteria, fungi, plants and mammalian systems including Homo sapiens. Despite its ubiquity and functional importance, the specific origins of this unique amino acid have eluded characterization. In this study, we describe the discovery and characterization of two enzyme pairs consisting of a pyridoxal 5'-phosphate (PLP)-linked aminotransferase and an unprecedented isomerase synergistically responsible for the biosynthesis of L-allo-Ile from L-isoleucine (L-Ile) in natural products. DsaD/DsaE from the desotamide biosynthetic pathway in Streptomyces scopuliridis SCSIO ZJ46, and MfnO/MfnH from the marformycin biosynthetic pathway in Streptomyces drozdowiczii SCSIO 10141 drive L-allo-Ile generation in each respective system. In vivo gene inactivations validated the importance of the DsaD/DsaE pair and MfnO/MfnH pair in L-allo-Ile unit biosynthesis. Inactivation of PLP-linked aminotransferases DsaD and MfnO led to significantly diminished desotamide and marformycin titers, respectively. Additionally, inactivation of the isomerase genes dsaE and mfnH completely abolished production of all L-allo-Ile-containing metabolites in both biosynthetic pathways. Notably, in vitro biochemical assays revealed that DsaD/DsaE and MfnO/MfnH each catalyze a bidirectional reaction between L-allo-Ile and L-Ile. Site-directed mutagenesis experiments revealed that the enzymatic reaction involves a PLP-linked ketimine intermediate and uses an arginine residue from the C-terminus of each isomerase to epimerize the amino acid β-position. Consequently, these data provide important new insight into the origins of L-allo-Ile in natural products with medicinal potential and illuminate new possibilities for biotool development.
Topics: Catalysis; Computational Biology; Humans; Isoleucine; Mutagenesis, Site-Directed
PubMed: 26669414
DOI: 10.1021/jacs.5b11380 -
Rapid Communications in Mass... Mar 2022The function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa,...
RATIONALE
The function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa, so the ability to identify the correct isomer using mass spectrometry can help resolve important biological questions. Tandem mass spectrometry approaches for Leu/Ile (Xle) discrimination have been developed, but they all have certain limitations.
METHODS
Four model peptides and two wild-type peptide sequences containing either Leu or Ile residues were subjected to charge transfer dissociation (CTD) mass spectrometry on a modified three-dimensional ion trap. The peptides were analyzed in both the 1+ and 2+ charge states, and the results were compared to conventional collision-induced dissociation spectra of the same peptides obtained using the same instrument.
RESULTS
CTD resulted in 100% sequence coverage for each of the studied peptides and provided a variety of side-chain cleavages, including d, w and v ions. Using CTD, reliable d and w ions of Xle residues were observed more than 80% of the time. When present, d ions are typically greater than 10% of the abundance of the corresponding a ions from which they derive, and w ions are typically more abundant than the z ions from which they derive.
CONCLUSIONS
CTD has the benefit of being applicable to both 1+ and 2+ precursor ions, and the overall performance is comparable to that of other high-energy activation techniques like hot electron capture dissociation and UV photodissociation. CTD does not require chemical modifications of the precursor peptides, nor does it require additional levels of isolation and fragmentation.
Topics: Amino Acid Sequence; Isoleucine; Leucine; Peptides; Tandem Mass Spectrometry
PubMed: 34927767
DOI: 10.1002/rcm.9246 -
Plant Physiology Aug 2022Plant organ abscission, a process that is important for development and reproductive success, is inhibited by the phytohormone auxin and promoted by another...
Plant organ abscission, a process that is important for development and reproductive success, is inhibited by the phytohormone auxin and promoted by another phytohormone, jasmonic acid (JA). However, the molecular mechanisms underlying the antagonistic effects of auxin and JA in organ abscission are unknown. We identified a tomato (Solanum lycopersicum) class III homeodomain-leucine zipper transcription factor, HOMEOBOX15A (SlHB15A), which was highly expressed in the flower pedicel abscission zone and induced by auxin. Knocking out SlHB15A using clustered regularly interspaced short palindromic repeats-associated protein 9 technology significantly accelerated abscission. In contrast, overexpression of microRNA166-resistant SlHB15A (mSlHB15A) delayed abscission. RNA sequencing and reverse transcription-quantitative PCR analyses showed that knocking out SlHB15A altered the expression of genes related to JA biosynthesis and signaling. Furthermore, functional analysis indicated that SlHB15A regulates abscission by depressing JA-isoleucine (JA-Ile) levels through inhabiting the expression of JASMONATE-RESISTANT1 (SlJAR1), a gene involved in JA-Ile biosynthesis, which could induce abscission-dependent and abscission-independent ethylene signaling. SlHB15A bound directly to the SlJAR1 promoter to silence SlJAR1, thus delaying abscission. We also found that flower removal enhanced JA-Ile content and that application of JA-Ile severely impaired the inhibitory effects of auxin on abscission. These results indicated that SlHB15A mediates the antagonistic effect of auxin and JA-Ile during tomato pedicel abscission, while auxin inhibits abscission through the SlHB15A-SlJAR1 module.
Topics: Cyclopentanes; Gene Expression Regulation, Plant; Indoleacetic Acids; Isoleucine; Solanum lycopersicum; Oxylipins; Plant Growth Regulators; Plant Proteins; Transcription Factors
PubMed: 35522030
DOI: 10.1093/plphys/kiac212