MALAT1 affects ovarian cancer cell behavior and patient survival

PubMed Central

Lin, Qunbo; Guan, Wencai; Ren, Weimin; Zhang, Lingyun; Zhang, Jinguo; Xu, Guoxiong

2018-01-01

Epithelial ovarian cancer (EOC) is one of the most lethal malignancies of the female reproductive organs. Increasing evidence has revealed that long non-coding RNAs (lncRNAs) participate in tumorigenesis. Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is an lncRNA and plays a role in various types of tumors. However, the function of MALAT1 on cellular behavior in EOC remains unclear. The current study explored the expression of MALAT1 in ovarian cancer tissues and in EOC cell lines. Quantitative RT-PCR analysis revealed that the expression of MALAT1 was higher in human ovarian malignant tumor tissues and EOC cells than in normal ovarian tissues and non-tumorous human ovarian surface epithelial cells, respectively. By analyzing the online database Kaplan-Meier Plotter, MALAT1 was identified to be correlated with the overall survival (OS) and progression-free survival (PFS) of patients with ovarian cancer. Furthermore, knockdown of MALAT1 by small interfering RNA (siRNA) significantly decreased EOC cell viability, migration, and invasion. Finally, dual-luciferase reporter assays demonstrated that MALAT1 interacted with miR-143-3p, a miRNA that plays a role in EOC as demonstrated in our previous study. Inhibition of MALAT1 resulted in an increase of miR-143-3p expression, leading to a decrease of CMPK protein expression. In conclusion, our results indicated that MALAT1 was overexpressed in EOC. Silencing of MALAT1 decreased EOC cell viability and inhibited EOC cell migration and invasion. These data revealed that MALAT1 may serve as a new therapeutic target of human EOC. PMID:29693187

Disruption of BCAA metabolism in mice impairs exercise metabolism and endurance.

PubMed

She, Pengxiang; Zhou, Yingsheng; Zhang, Zhiyou; Griffin, Kathleen; Gowda, Kavitha; Lynch, Christopher J

2010-04-01

Exercise enhances branched-chain amino acid (BCAA) catabolism, and BCAA supplementation influences exercise metabolism. However, it remains controversial whether BCAA supplementation improves exercise endurance, and unknown whether the exercise endurance effect of BCAA supplementation requires catabolism of these amino acids. Therefore, we examined exercise capacity and intermediary metabolism in skeletal muscle of knockout (KO) mice of mitochondrial branched-chain aminotransferase (BCATm), which catalyzes the first step of BCAA catabolism. We found that BCATm KO mice were exercise intolerant with markedly decreased endurance to exhaustion. Their plasma lactate and lactate-to-pyruvate ratio in skeletal muscle during exercise and lactate release from hindlimb perfused with high concentrations of insulin and glucose were significantly higher in KO than wild-type (WT) mice. Plasma and muscle ammonia concentrations were also markedly higher in KO than WT mice during a brief bout of exercise. BCATm KO mice exhibited 43-79% declines in the muscle concentration of alanine, glutamine, aspartate, and glutamate at rest and during exercise. In response to exercise, the increments in muscle malate and alpha-ketoglutarate were greater in KO than WT mice. While muscle ATP concentration tended to be lower, muscle IMP concentration was sevenfold higher in KO compared with WT mice after a brief bout of exercise, suggesting elevated ammonia in KO is derived from the purine nucleotide cycle. These data suggest that disruption of BCAA transamination causes impaired malate/aspartate shuttle, thereby resulting in decreased alanine and glutamine formation, as well as increases in lactate-to-pyruvate ratio and ammonia in skeletal muscle. Thus BCAA metabolism may regulate exercise capacity in mice.

Expression of long noncoding RNA MALAT1 correlates with increased levels of Nischarin and inhibits oncogenic cell functions in breast cancer.

PubMed

Eastlack, Steven C; Dong, Shengli; Mo, Yin Y; Alahari, Suresh K

2018-01-01

Malat1 is a long noncoding RNA with a wide array of functions, including roles in regulating cancer cell migration and metastasis. However, the nature of its involvement in control of these oncogenic processes is incompletely understood. In the present study, we investigate the role of Malat1 and the effects of Malat1 KO in a breast cancer cell model. Our selection of Malat1 as the subject of inquiry followed initial screening experiments seeking to identify lncRNAs which are altered in the presence or absence of Nischarin, a gene of interest previously discovered by our lab. Nischarin is a well characterized tumor suppressor protein and actively represses cell proliferation, migration, and invasion in breast cancer. Our microarray screen for lncRNAs revealed multiple lncRNAs to be significantly elevated in cells ectopically expressing Nischarin compared to control cancer cells, which have only marginal Nisch expression. Using these cells, we assess how the link between Nischarin and Malat1 affects cancer cell function, finding that Malat1 confers an inhibitory effect on cell growth and migration which is lost following Malat1 KO, but in a Nisch-dependent context. Specifically, Malat1 KO in the background of low Nischarin expression had a limited effect on cell functions, while Malat1 KO in cells with high levels of Nischarin led to significant increases in cell proliferation and migration. In summary, this project provides further clarity concerning the function of Malat1, specifically in breast cancer, while also indicating that the Nischarin expression context is an important factor in the determining how Malat1 activity is governed in breast cancer.

Long noncoding RNA MALAT1 regulates generation of reactive oxygen species and the insulin responses in male mice.

PubMed

Chen, Jingshu; Ke, Sui; Zhong, Lei; Wu, Jing; Tseng, Alexander; Morpurgo, Benjamin; Golovko, Andrei; Wang, Gang; Cai, James J; Ma, Xi; Li, Defa; Tian, Yanan

2018-06-01

The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA and its overexpression is associated with the development of many types of malignancy. MALAT1 null mice show no overt phenotype. However, in transcriptome analysis of MALAT1 null mice we found significant upregulation of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulated antioxidant genes including Nqo1 and Cat with significant reduction in reactive oxygen species (ROS) and greatly reduced ROS-generated protein carbonylation in hepatocyte and islets. We performed lncRNA pulldown assay using biotinylated antisense oligonucleotides against MALAT1 and found MALAT1 interacted with Nrf2, suggesting Nrf2 is transcriptionally regulated by MALAT1. Exposure to excessive ROS has been shown to cause insulin resistance through activation of c-Jun N-terminal kinase (JNK) which leads to inhibition of insulin receptor substrate 1 (IRS-1) and insulin-induced phosphorylation of serine/threonine kinase Akt. We found MALAT1 ablation suppressed JNK activity with concomitant insulin-induced activation of IRS-1 and phosphorylation of Akt suggesting MALAT1 regulated insulin responses. MALAT1 null mice exhibited sensitized insulin-signaling response to fast-refeeding and glucose/insulin challenges and significantly increased insulin secretion in response to glucose challenge in isolated MALAT1 null islets, suggesting an increased insulin sensitivity. In summary, we demonstrate that MALAT1 plays an important role in regulating insulin sensitivity and has the potential as a therapeutic target for the treatment of diabetes as well as other diseases caused by excessive exposure to ROS. Copyright © 2018. Published by Elsevier Inc.

Are phloem-derived amino acids the origin of the elevated malate concentration in the xylem sap following mineral N starvation in soybean?

PubMed

Vitor, Simone C; do Amarante, Luciano; Sodek, Ladaslav

2018-05-16

A substantial increase in malate in the xylem sap of soybean subjected to mineral N starvation originates mainly from aspartate, a prominent amino acid of the phloem. A substantial increase in xylem malate was found when non-nodulated soybean plants were transferred to a N-free medium. Nodulated plants growing in the absence of mineral N and, therefore, dependent on symbiotic N 2 fixation also contained elevated concentrations of malate in the xylem sap. When either nitrate or ammonium was supplied, malate concentrations in the xylem sap were low, both for nodulated and non-nodulated plants. Evidence was obtained that the elevated malate concentration of the xylem was derived from amino acids supplied by the phloem. Aspartate was a prominent component of the phloem sap amino acids and, therefore, a potential source of malate. Supplying the roots of intact plants with 13 C-aspartate revealed that malate of the xylem sap was readily labelled under N starvation. A hypothetical scheme is proposed whereby aspartate supplied by the phloem is metabolised in the roots and the products of this metabolism cycled back to the shoot. Under N starvation, aspartate metabolism is diverted from asparagine synthesis to supply N for the synthesis of other amino acids via transaminase activity. The by-product of aspartate transaminase activity, oxaloacetate, is transformed to malate and its export accounts for much of the elevated concentration of malate found in the xylem sap. This mechanism represents a new additional role for malate during mineral N starvation of soybean, beyond that of charge balance.

Effects of L-malate on physical stamina and activities of enzymes related to the malate-aspartate shuttle in liver of mice.

PubMed

Wu, J L; Wu, Q P; Huang, J M; Chen, R; Cai, M; Tan, J B

2007-01-01

L-malate, a tricarboxylic acid cycle (TCA) intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production and may be involved in the beneficial effects of improving physical stamina. In the present study, we investigated the effects of L-malate on the performance of forced swimming time and blood biochemical parameters related to fatigue - blood urea nitrogen (BUN), glucose (Glc), creatine kinase (CK),total protein (TP) and lactic acid (LA). To investigate the effects of L-malate on the malate-aspartate shuttle and energy metabolism in mice, the activities of enzymes related to the malate-aspartate shuttle were measured. L-malate was orally administered to mice continuously for 30 days using a feeding atraumatic needle. The swimming time was increased by 26.1 % and 28.5 %, respectively, in the 0.210 g/kg and 0.630 g/kg L-malate-treated group compared with the control group. There were no differences in the concentrations of Glc, BUN and TP between the L-malate-treated groups and the control groups. However, the levels of CK were significantly decreased in the L-malate-treated groups. The results predict a potential benefit of L-malate for improving physical stamina and minimizing muscle damage during swimming exercise. The activities of cytosolic and mitochondrial malate dehydrogenase were significantly elevated in the L-malate-treated group compared with the control group. These enzymatic activities may be useful indicators for evaluating changes affecting the malate-aspartate shuttle and energy metabolism in the liver of mice.

Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase.

PubMed

Taillefer, M; Rydzak, T; Levin, D B; Oresnik, I J; Sparling, R

2015-04-01

Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a kcat of 45.8 s(-1) or 14.9 s(-1), respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a kcat of 520.8 s(-1). The malic enzyme used NADP(+) as a cofactor along with NH4 (+) and Mn(2+) as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a Ki of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4 (+) based on the characterization of the malate dehydrogenase and malic enzyme. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Oxaloacetate enhances neuronal cell bioenergetic fluxes and infrastructure.

PubMed

Wilkins, Heather M; Koppel, Scott; Carl, Steven M; Ramanujan, Suruchi; Weidling, Ian; Michaelis, Mary L; Michaelis, Elias K; Swerdlow, Russell H

2016-04-01

We tested how the addition of oxaloacetate (OAA) to SH-SY5Y cells affected bioenergetic fluxes and infrastructure, and compared the effects of OAA to malate, pyruvate, and glucose deprivation. OAA displayed pro-glycolysis and pro-respiration effects. OAA pro-glycolysis effects were not a consequence of decarboxylation to pyruvate because unlike OAA, pyruvate lowered the glycolysis flux. Malate did not alter glycolysis flux and reduced mitochondrial respiration. Glucose deprivation essentially eliminated glycolysis and increased mitochondrial respiration. OAA increased, while malate decreased, the cell NAD+/NADH ratio. Cytosolic malate dehydrogenase 1 protein increased with OAA treatment, but not with malate or glucose deprivation. Glucose deprivation increased protein levels of ATP citrate lyase, an enzyme which produces cytosolic OAA, whereas OAA altered neither ATP citrate lyase mRNA nor protein levels. OAA, but not glucose deprivation, increased cytochrome oxidase subunit 2, PGC1α, PGC1β, and PGC1 related co-activator protein levels. OAA increased total and phosphorylated SIRT1 protein. We conclude that adding OAA to SH-SY5Y cells can support or enhance both glycolysis and respiration fluxes. These effects appear to depend, at least partly, on OAA causing a shift in the cell redox balance to a more oxidized state, that it is not a glycolysis pathway intermediate, and possibly its ability to act in an anaplerotic fashion. We examined how oxaloacetate (OAA) affects bioenergetic fluxes. To advance the understanding of how OAA mediates these changes, we compared the effects of OAA to malate, pyruvate, and glucose deprivation. We further examined how OAA affects levels of enzymes that facilitate its cytosolic metabolism, and found OAA increased the expression of malate dehydrogenase 1 (MDH1-cytosolic). We propose the following: OAA supports both glycolysis and respiration fluxes, shifts the cell redox balance toward a more oxidized state, and acts in an anaplerotic fashion. Abbreviations not defined in the text: MDH2, malate dehydrogenase 2 (mitochondrial). © 2016 International Society for Neurochemistry.

Metabolic engineering of Aspergillus oryzae for efficient production of l-malate directly from corn starch.

PubMed

Liu, Jingjing; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian; Liu, Long

2017-11-20

l-Malate, an important chemical building block, has been widely applied in the food, pharmaceutical, and bio-based materials industries. In previous work, we engineered Aspergillus oryzae by rewiring the reductive tricarboxylic acid pathway to produce l-malate from glucose. To decrease the production cost, here, we further engineered A. oryzae to efficiently produce l-malate directly from corn starch with simultaneous liquefaction-saccharification and fermentation. First, a promoter PN5 was constructed by quintuple tandem of the 97-bp fragment containing the cis-element of the glucoamylase gene promoter (PglaA), and with the promoter PN5, the transcriptional level of glaA gene increased by 25-45%. Then, by co-overexpression of glaA, a-amylase (amyB) and a-glucosidase (agdA) genes with the promoter PN5, the l-malate titer increased from 55.5g/L to 72.0g/L with 100g/L corn starch in shake flask. In addition, to reduce the concentration of byproducts succinate and fumarate, a fumarase from Saccharomyces cerevisiae, which facilitated the transformation of fumarate to l-malate, was overexpressed. As a result, the concentration of succinate and fumarate decreased from 12.6 and 1.29g/L to 7.8 and 0.59g/L, and the l-malate titer increased from 72.0g/L to 78.5g/L. Finally, we found that the addition of glucose at the initial fermentation stage facilitated the cell growth and l-malate synthesis, and the l-malate titer further increased to 82.3g/L by co-fermentation of 30g/L glucose and 70g/L corn starch, with a productivity of 1.18g/L/h and a yield of 0.82g/g total carbon sources. Copyright © 2017 Elsevier B.V. All rights reserved.

Altered Expression of a Malate-Permeable Anion Channel, OsALMT4, Disrupts Mineral Nutrition1[OPEN

PubMed Central

Delhaize, Emmanuel

2017-01-01

Aluminum-activated malate transporters (ALMTs) form a family of anion channels in plants, but little is known about most of its members. This study examined the function of OsALMT4 from rice (Oryza sativa). We show that OsALMT4 is expressed in roots and shoots and that the OsALMT4 protein localizes to the plasma membrane. Transgenic rice lines overexpressing (OX) OsALMT4 released malate from the roots constitutively and had 2-fold higher malate concentrations in the xylem sap than nulls, indicating greater concentrations of malate in the apoplast. OX lines developed brown necrotic spots on the leaves that did not appear on nulls. These symptoms were not associated with altered concentrations of any mineral element in the leaves, although the OX lines had higher concentrations of Mn and B in their grain compared with nulls. While total leaf Mn concentrations were not different between the OX and null lines, Mn concentrations in the apoplast were greater in the OX plants. The OX lines also displayed increased expression of Mn transporters and were more sensitive to Mn toxicity than null plants. We showed that the growth of wild-type rice was unaffected by 100 µm Mn in hydroponics but, when combined with 1 mm malate, this concentration inhibited growth. We conclude that increasing OsALMT4 expression affected malate efflux and compartmentation within the tissues, which increased Mn concentrations in the apoplast of leaves and induced the toxicity symptoms. This study reveals new links between malate transport and mineral nutrition. PMID:29101278

Long noncoding RNA MALAT1 promotes osterix expression to regulate osteogenic differentiation by targeting miRNA-143 in human bone marrow-derived mesenchymal stem cells.

PubMed

Gao, Yuan; Xiao, Fei; Wang, Chenglong; Wang, Chuandong; Cui, Penglei; Zhang, Xiaoling; Chen, Xiaodong

2018-05-09

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is essential for the human bone formation, and emerging evidence shows that long non-coding RNAs (lncRNAs) play important roles in hBMSC osteogenic differentiation. MALAT1 is often regarded as a tumor-related lncRNA, but its function in mesenchymal stem cell differentiation remains to be defined. In this study, we aimed to investigate whether MALAT1 regulates Osterix (Osx) expression by sponging miR-143 to promote hBMSC osteogenic differentiation. Firstly, we found that the expression of MALAT1 was much lower in hBMSCs from osteoporosis patients and miR-143 was contrarily higher. In addition, MALAT1 expression increased, and miR-143 decreased when hBMSCs were treated with osteogenic induction. Then, we used short hairpin RNAs to knockdown MALAT1, and the results showed that hBMSC osteogenic differentiation decreased significantly, indicating that MALAT1 is a positive regulator of osteogenic differentiation in hBMSCs. Furthermore, by luciferase assays, we found that MALAT1 could directly bind to miR-143 and negatively regulate its expression. Similarly, miR-143 could directly bind to the target site on the Osx 3'-UTR and then inhibit Osx expression. Knockdown of MALAT1 decreased Osx expression, and co-transfection of miR-143 inhibitor could rescue Osx mRNA expression. While Osx expression was increased in MALAT1-overexpressing hBMSCs, it was reversed by the miR-143 mimics. Moreover, Osx silencing decreased ALP, OCN, and OPN mRNA expression induced by the miR-143 inhibitor. Altogether, our findings suggest that MALAT1 acts to regulate Osx expression through targeting miR-143; thus, it is considered as a positive regulator in hBMSC osteogenic differentiation. © 2018 Wiley Periodicals, Inc.

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

PubMed

Hecht, K; Wrba, A; Jaenicke, R

1989-07-15

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

MALAT1 predicts poor survival in osteosarcoma patients and promotes cell metastasis through associating with EZH2

PubMed Central

Huo, Yanqing; Li, Qingbo; Wang, Xiqian; Jiao, Xiejia; Zheng, Jiachun; Li, Zhiqiang; Pan, Xiaohan

2017-01-01

Osteosarcoma is the most common type of bone cancer, especially in children and young adults. Recently, long noncoding RNAs (lncRNAs) have emerged as new prognostic markers and gene regulators in several cancers, including osteosarcoma. In this study, we investigated the contributions of the lncRNA MALAT1 in osteosarcoma with a specific focus on its transcriptional regulation and its interaction with EZH2. Our results showed that MALAT1 was significantly increased in osteosarcoma specimens and cell lines. ROC curve analysis showed that MALAT1 had a higher area under the curve than alkaline phosphatase, and Kaplan-Meier survival analysis indicated that patients with high serum levels of MALAT1 showed reduced survival rate. Knockdown of MALAT1 decreased osteosarcoma cell invasion and promoted E-cadherin expression. Mechanistic investigations showed that MALAT1 was transcriptionally activated by TGF-β. Additionally, EZH2 is highly expressed and associated with the 3’ end region of lncRNA MALAT1 in osteosarcoma, and this association finally suppressed the expression of E-cadherin. Subsequently, our gain and loss function assay showed that MALAT1 overexpression promoted cell metastasis and decreased E-cadherin level, however, this effect was partially reversed by EZH2 knockdown. In conclusion, our work illuminates that lncRNA MALAT1 is a potential diagnostic and prognostic factor in osteosarcoma and further demonstrates how MALAT1 confers an oncogenic function. Thus, lncRNA MALAT1 may serve as a promising prognostic and therapeutic target for osteosarcoma patients. PMID:28388584

Skeletal Muscle Acute and Chronic Metabolic Response to Essential Amino Acid Supplementation in Hypertriglyceridemic Older Adults

PubMed Central

Marquis, Bryce J; Hurren, Nicholas M; Carvalho, Eugenia; Kim, Il-Young; Schutzler, Scott; Azhar, Gohar; Wolfe, Robert R; Børsheim, Elisabet

2017-01-01

Abstract Background: Supplementation with essential amino acids (EAAs) + arginine is a promising nutritional approach to decrease plasma triglyceride (TG) concentrations, which are an independent risk factor for ischemic heart disease. Objective: The objective of this study was to examine the effects of 8 wk of EAA supplementation on skeletal muscle basal metabolite concentrations and changes in metabolic response to acute EAA intake, with an emphasis on mitochondrial metabolism, in adults with elevated TGs to better understand the mechanisms of lowering plasma TGs. Methods: Older adults with elevated plasma TG concentrations were given 22 g EAAs to ingest acutely before and after an 8-wk EAA supplementation period. Skeletal muscle biopsy samples were collected before and after acute EAA intake, both pre- and postsupplementation (4 biopsy samples), and targeted metabolomic analyses of organic acids and acylcarnitines were conducted on the specimens. Results: Acute EAA intake resulted in increased skeletal muscle acylcarnitine concentrations associated with oxidative catabolism of the supplement components, with the largest increases found in acylcarnitines of branched-chain amino acid oxidative catabolism, including isovaleryl-carnitine (2200%) and 2-methylbutyryl-carnitine (2400%). The chronic EAA supplementation resulted in a 19% decrease in plasma TGs along with accumulation of long-chain acylcarnitines myristoyl- (90%) and stearoyl- (120%) carnitine in skeletal muscle and increases in succinyl-carnitine (250%) and the late-stage tricarboxylic acid cycle intermediates fumarate (44%) and malate (110%). Conclusions: Supplementation with EAAs shows promise as an approach for moderate reduction in plasma TGs. Changes in skeletal muscle metabolites suggest incomplete fatty acid oxidation and increased anaplerosis, which suggests a potential bottleneck in fatty acid metabolism.

An InDel in the Promoter of Al-ACTIVATED MALATE TRANSPORTER9 Selected during Tomato Domestication Determines Fruit Malate Contents and Aluminum Tolerance[OPEN

PubMed Central

Wang, Xin; Hu, Tixu; Zhang, Fengxia; Wang, Bing; Li, Changxin; Yang, Tianxia; Li, Hanxia; Lu, Yongen; Ye, Zhibiao

2017-01-01

Deciphering the mechanism of malate accumulation in plants would contribute to a greater understanding of plant chemistry, which has implications for improving flavor quality in crop species and enhancing human health benefits. However, the regulation of malate metabolism is poorly understood in crops such as tomato (Solanum lycopersicum). Here, we integrated a metabolite-based genome-wide association study with linkage mapping and gene functional studies to characterize the genetics of malate accumulation in a global collection of tomato accessions with broad genetic diversity. We report that TFM6 (tomato fruit malate 6), which corresponds to Al-ACTIVATED MALATE TRANSPORTER9 (Sl-ALMT9 in tomato), is the major quantitative trait locus responsible for variation in fruit malate accumulation among tomato genotypes. A 3-bp indel in the promoter region of Sl-ALMT9 was linked to high fruit malate content. Further analysis indicated that this indel disrupts a W-box binding site in the Sl-ALMT9 promoter, which prevents binding of the WRKY transcription repressor Sl-WRKY42, thereby alleviating the repression of Sl-ALMT9 expression and promoting high fruit malate accumulation. Evolutionary analysis revealed that this highly expressed Sl-ALMT9 allele was selected for during tomato domestication. Furthermore, vacuole membrane-localized Sl-ALMT9 increases in abundance following Al treatment, thereby elevating malate transport and enhancing Al resistance. PMID:28814642

Role of malate transporter in lipid accumulation of oleaginous fungus Mucor circinelloides.

PubMed

Zhao, Lina; Cánovas-Márquez, José T; Tang, Xin; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

2016-02-01

Fatty acid biosynthesis in oleaginous fungi requires the supply of reducing power, NADPH, and the precursor of fatty acids, acetyl-CoA, which is generated in the cytosol being produced by ATP: citrate lyase which requires citrate to be, transported from the mitochondrion by the citrate/malate/pyruvate transporter. This transporter, which is within the mitochondrial membrane, transports cytosolic malate into the mitochondrion in exchange for mitochondrial citrate moving into the cytosol (Fig. 1). The role of malate transporter in lipid accumulation in oleaginous fungi is not fully understood, however. Therefore, the expression level of the mt gene, coding for a malate transporter, was manipulated in the oleaginous fungus Mucor circinelloides to analyze its effect on lipid accumulation. The results showed that mt overexpression increased the lipid content for about 70 % (from 13 to 22 % dry cell weight, CDW), whereas the lipid content in mt knockout mutant decreased about 27 % (from 13 to 9.5 % CDW) compared with the control strain. Furthermore, the extracellular malate concentration was decreased in the mt overexpressing strain and increased in the mt knockout strain compared with the wild-type strain. This work suggests that the malate transporter plays an important role in regulating lipid accumulation in oleaginous fungus M. circinelloides.

Expression of metastasis-associated lung adenocarcinoma transcript 1 long non-coding RNA in vitro and in patients with non-small cell lung cancer.

PubMed

Lin, Ling; Li, Haiyan; Zhu, Yefei; He, Susu; Ge, Hongfei

2018-06-01

The present study aimed to investigate the association between the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long non-coding RNA (lncRNA) and the recurrence of non-small cell lung cancer (NSCLC) and to elucidate the potential mechanisms of MALAT1 in vitro . Between 1 June 1, 2010 and December 30, 2016, NSCLC tumor tissues and adjacent non-cancerous tissues were obtained from 120 patients with NSCLC, who had undergone surgical resection at Taizhou Hospital of Wenzhou Medical University (Linhai, China). The total RNA of tissues and cells were extracted and the expression of MALAT1 was determined using a wound healing assay and reverse transcription quantitative polymerase chain reaction. In addition, MALAT1 expression in A549 cells was silenced using small interfering RNA. The proliferation, migration and invasion of cells were then assessed using a CellTiter 96 kit and Transwell assays. MALAT1 expression was significantly increased in NSCLC samples compared with expression in adjacent non-cancerous tissues. Furthermore, the expression of MALAT1 in patients with NSCLC that exhibited recurrence was markedly higher than in those that did not. The results of the present study also demonstrated significant associations between high expression of MALAT1 and female sex, Tumor-Node-Metastasis advanced stage, vessel invasion, pathological differentiation and recurrence of patients with NSCLC. The proliferative, migratory and invasive abilities of MALAT1-silenced A549 cells were significantly decreased compared with those of control cells. MALAT1 expression was significantly increased in NSCLC tissues and was revealed to serve a role in the progression of NSCLC.

Oxaloacetate Enhances Neuronal Cell Bioenergetic Fluxes and Infrastructure

PubMed Central

Wilkins, Heather M.; Koppel, Scott; Carl, Steven M.; Ramanujan, Suruchi; Weidling, Ian; Michaelis, Mary L.; Michaelis, Elias K.; Swerdlow, Russell H.

2017-01-01

We tested how the addition of oxaloacetate (OAA) to SH-SY5Y cells affected bioenergetic fluxes and infrastructure, and compared the effects of OAA to malate, pyruvate, and glucose deprivation. OAA displayed pro-glycolysis and pro-respiration effects. OAA pro-glycolysis effects were not a consequence of decarboxylation to pyruvate because unlike OAA, pyruvate lowered the glycolysis flux. Malate did not alter glycolysis flux and reduced mitochondrial respiration. Glucose deprivation essentially eliminated glycolysis and increased mitochondrial respiration. OAA increased, while malate decreased, the cell NAD+/NADH ratio. Cytosolic malate dehydrogenase 1 (MDH1) protein increased with OAA treatment, but not with malate or glucose deprivation. Glucose deprivation increased protein levels of ATP citrate lyase, an enzyme which produces cytosolic OAA, while OAA altered neither ATP citrate lyase mRNA nor protein levels. OAA, but not glucose deprivation, increased COX2, PGC1α, PGC1β, and PRC protein levels. OAA increased total and phosphorylated SIRT1 protein. We conclude that adding OAA to SH-SY5Y cells can support or enhance both glycolysis and respiration fluxes. These effects appear to depend, at least partly, on OAA causing a shift in the cell redox balance to a more oxidized state, that it is not a glycolysis pathway intermediate, and possibly its ability to act in an anaplerotic fashion. PMID:26811028

Day-night variations in malate concentration, osmotic pressure, and hydrostatic pressure in Cereus validus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luettge, U.; Nobel, P.S.

1984-07-01

Malate concentration and stem osmotic pressure concomitantly increase during nighttime CO/sub 2/ fixation and then decrease during the daytime in the obligate Crassulacean acid metabolism (CAM) plant, Cereus validus (Cactaceae). Changes in malate osmotic pressure calculated using the Van't Hoff relation match the changes in stem osmotic pressure, indicating that changes in malate level affected the water relations of the succulent stems. In contrast to stem osmotic pressure, stem water potential showed little day-night changes, suggesting that changes in cellular hydrostatic pressure occurred. This was corroborated by direct measurements of hydrostatic pressure using the Juelich pressure probe where a smallmore » oil-filled micropipette is inserted directly into chlorenchyma cells, which indicated a 4-fold increase in hydrostatic pressure from dusk to dawn. A transient increase of hydrostatic pressure at the beginning of the dark period was correlated with a short period of stomatal closing between afternoon and nighttime CO/sub 2/ fixation, suggesting that the rather complex hydrostatic pressure patterns could be explained by an interplay between the effects of transpiration and malate levels. A second CAM plant, Agave deserti, showed similar day-night changes in hydrostatic pressure in its succulent leaves. It is concluded that, in addition to the inverted stomatal rhythm, the oscillations of malate markedly affect osmotic pressures and hence water relations of CAM plants. 13 references, 4 figures.« less

High expression of long non-coding RNA MALAT1 in breast cancer is associated with poor relapse-free survival.

PubMed

Wang, Zhanwei; Katsaros, Dionyssios; Biglia, Nicoletta; Shen, Yi; Fu, Yuanyuan; Loo, Lenora W M; Jia, Wei; Obata, Yuki; Yu, Herbert

2018-05-29

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been identified as a prognostic marker for the metastasis of early-stage non-small cell lung cancer (NSCLCs). We studied MALAT1 expression in breast cancer in relation to disease features and patient survival. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to measure MALAT1 expression in tumor samples of 509 breast cancer patients. Hazards ratios (HRs) and 95% confidence intervals (CIs) were calculated to assess the association between MALAT1 expression and breast cancer survival using the Cox proportional hazards regression model, and the analysis was adjusted for age at surgery, tumor grade, disease stage, and hormone receptor status. Meta-analysis of multiple microarray datasets from online databases and our own study was performed to evaluate the association of MALAT1 with breast cancer survival. Patients with low-grade or ER-positive tumors had higher expression of MALAT1 compared to those with high-grade (p = 0.013) or ER-negative (p = 0.0002) tumors. Patients with PR-positive tumors also had higher MALAT1 expression than those with PR-negative tumors (p < 0.0001). In patients with positive hormone receptors or low tumor grade, tumors with high MALAT1 expression were more likely to recur. Survival analysis showed that patients with high expression of MALAT1 had a twofold increase in risk of relapse (p = 0.0083) compared to those with low expression. This association remained significant after adjustment for age at surgery, disease stage, tumor grade, and hormone receptor status. Meta-analysis showed that high MALAT1 expression was associated with poor relapse-free survival in patients with hormone receptor-positive tumors (HR 1.44, 95% CI 1.08-1.92). High expression of lncRNA MALAT1 is associated with breast cancer relapse and may play a role in tumor progression.

Oxaloacetate and malate production in engineered Escherichia coli by expression of codon-optimized phosphoenolpyruvate carboxylase2 gene from Dunaliella salina.

PubMed

Park, Soohyun; Chang, Kwang Suk; Jin, Eonseon; Pack, Seung Pil; Lee, Jinwon

2013-01-01

A new phosphoenolpyruvate carboxylase (PEPC) gene of Dunaliella salina is identified using homology analysis was conducted using PEPC gene of Chlamydomonas reinhardtii and Arabidopsis thaliana. Recombinant E. coli SGJS115 with increased production of malate and oxaloacetate was developed by introducing codon-optimized phosphoenolpyruvate carboxylase2 (OPDSPEPC2) gene of Dunaliella salina. E. coli SGJS115 yielded a 9.9 % increase in malate production. In addition, E. coli SGJS115 exhibited two times increase in the yield of oxaloacetate over the E. coli SGJS114 having identified PEPC2 gene obtained from Dunaliella salina.

BoALMT1, an Al-Induced Malate Transporter in Cabbage, Enhances Aluminum Tolerance in Arabidopsis thaliana

PubMed Central

Zhang, Lei; Wu, Xin-Xin; Wang, Jinfang; Qi, Chuandong; Wang, Xiaoyun; Wang, Gongle; Li, Mingyue; Li, Xingsheng; Guo, Yang-Dong

2018-01-01

Aluminum (Al) is present in approximately 50% of the arable land worldwide and is regarded as the main limiting factor of crop yield on acidic soil. Al-induced root malate efflux plays an important role in the Al tolerance of plants. Here, the aluminum induced malate transporter BoALMT1 (KF322104) was cloned from cabbage (Brassica oleracea). BoALMT1 showed higher expression in roots than in shoots. The expression of BoALMT1 was specifically induced by Al treatment, but not the trivalent cations lanthanum (La), cadmium (Cd), zinc (Zn), or copper (Cu). Subcellular localization studies were performed in onion epidermal cells and revealed that BoALMT1 was localized at the plasma membrane. Scanning Ion-selective Electrode Technique was used to analyze H+ flux. Xenopus oocytes and Arabidopsis thaliana expressing BoALMT1 excreted more H+ under Al treatment. Overexpressing BoALMT1 in transgenic Arabidopsis resulted in enhanced Al tolerance and increased malate secretion. The results suggested that BoALMT1 functions as an Al-resistant gene and encodes a malate transporter. Expressing BoALMT1 in Xenopus oocytes or A. thaliana indicated that BoALMT1 could increase malate secretion and H+ efflux to resist Al tolerance. PMID:29410672

BoALMT1, an Al-Induced Malate Transporter in Cabbage, Enhances Aluminum Tolerance in Arabidopsis thaliana.

PubMed

Zhang, Lei; Wu, Xin-Xin; Wang, Jinfang; Qi, Chuandong; Wang, Xiaoyun; Wang, Gongle; Li, Mingyue; Li, Xingsheng; Guo, Yang-Dong

2017-01-01

Aluminum (Al) is present in approximately 50% of the arable land worldwide and is regarded as the main limiting factor of crop yield on acidic soil. Al-induced root malate efflux plays an important role in the Al tolerance of plants. Here, the aluminum induced malate transporter BoALMT1 (KF322104) was cloned from cabbage ( Brassica oleracea ). BoALMT1 showed higher expression in roots than in shoots. The expression of BoALMT1 was specifically induced by Al treatment, but not the trivalent cations lanthanum (La), cadmium (Cd), zinc (Zn), or copper (Cu). Subcellular localization studies were performed in onion epidermal cells and revealed that BoALMT1 was localized at the plasma membrane. Scanning Ion-selective Electrode Technique was used to analyze H + flux. Xenopus oocytes and Arabidopsis thaliana expressing BoALMT1 excreted more H + under Al treatment. Overexpressing BoALMT1 in transgenic Arabidopsis resulted in enhanced Al tolerance and increased malate secretion. The results suggested that BoALMT1 functions as an Al-resistant gene and encodes a malate transporter. Expressing BoALMT1 in Xenopus oocytes or A. thaliana indicated that BoALMT1 could increase malate secretion and H+ efflux to resist Al tolerance.

Enhancement of malate-production and increase in sensitivity to dimethyl succinate by mutation of the VID24 gene in Saccharomyces cerevisiae.

PubMed

Negoro, Hiroaki; Kotaka, Atsushi; Matsumura, Kengo; Tsutsumi, Hiroko; Hata, Yoji

2016-06-01

Malate in sake (a Japanese alcoholic beverage) is an important component for taste that is produced by yeasts during alcoholic fermentation. To date, many researchers have developed methods for breeding high-malate-producing yeasts; however, genes responsible for the high-acidity phenotype are not known. We determined the mutated gene involved in high malate production in yeast, isolated as a sensitive mutant to dimethyl succinate. In the comparative whole genome analysis between high-malate-producing strain and its parent strain, one of the non-synonymous substitutions was identified in the VID24 gene. The mutation of VID24 resulted in enhancement of malate-productivity and sensitivity to dimethyl succinate. The mutation appeared to lead to a deficiency in Vid24p function. Furthermore, disruption of cytoplasmic malate dehydrogenase (Mdh2p) gene in the VID24 mutant inhibited the high-malate-producing phenotype. Vid24p is known as a component of the multisubunit ubiquitin ligase and participates in the degradation of gluconeogenic enzymes such as Mdh2p. We suggest that the enhancement of malate-productivity results from an accumulation of Mdh2p due to the loss of Vid24p function. These findings propose a novel mechanism for the regulation of organic acid production in yeast cells by the component of ubiquitin ligase, Vid24p. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Mitochondria from the left heart ventricles of both normotensive and spontaneously hypertensive rats oxidize externally added NADH mostly via a novel malate/oxaloacetate shuttle as reconstructed in vitro.

PubMed

Atlante, Anna; Seccia, Teresa M; De Bari, Lidia; Marra, Ersilia; Passarella, Salvatore

2006-07-01

A substantial increase in NADH production, arising from accelerated glycolysis, occurs in cardiac hypertrophy and this raises the question of how the NADH is oxidised. We have addressed this problem by reconstructing appropriate mitochondrial shuttles in vitro, using mitochondria from the left ventricles of both normotensive and spontaneously hypertensive rats at 5 and 24 weeks of age as model systems for left ventricle hypertrophy and hypertrophy/hypertension respectively. We found that most NADH oxidation occurs via a novel malate/oxaloacetate shuttle, the activity of which increases with time and with the progression of hypertrophy and development of hypertension as judged by statistical ANOVA analysis. In contrast, alpha-glycerol-phosphate and the malate/aspartate shuttles were shown to make only a minor contribution to NADH oxidation in a manner essentially independent of age and progression of hypertrophy/hypertension. The rate of malate transport in exchange with oxaloacetate proved to limit the rate of NADH oxidation via this malate/oxaloacetate shuttle.

The Role of Vacuolar Malate-Transport Capacity in Crassulacean Acid Metabolism and Nitrate Nutrition. Higher Malate-Transport Capacity in Ice Plant after Crassulacean Acid Metabolism-Induction and in Tobacco under Nitrate Nutrition1

PubMed Central

Lüttge, Ulrich; Pfeifer, Tanja; Fischer-Schliebs, Elke; Ratajczak, Rafael

2000-01-01

Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H+-transport by H+-pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, ΔμH+, across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, Jmalrel, of apparent ATP-dependent malate-stimulated H+-transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. Jmalrel was high in the obligate crassulacean acid metabolism (CAM) species Kalanchoë daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO3− nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher Jmalrel. It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO3− nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins. PMID:11080309

Expression of novel cytosolic malate dehydrogenases (cMDH) in Lupinus angustifolius nodules during phosphorus starvation.

PubMed

Le Roux, Marcellous; Phiri, Ethel; Khan, Wesaal; Sakiroğlu, Muhammet; Valentine, Alex; Khan, Sehaam

2014-11-01

During P deficiency, the increased activity of malate dehydrogenase (MDH, EC 1.1.1.37) can lead to malate accumulation. Cytosolic- and nodule-enhanced MDH (cMDH and neMDH, respectively) are known isoforms, which contribute to MDH activity in root nodules. The aim of this study was to investigate the role of the cMDH isoforms in nodule malate supply under P deficiency. Nodulated lupins (Lupinus angustifolius var. Tanjil) were hydroponically grown at adequate P (+P) or low P (-P). Total P concentration in nodules decreased under P deficiency, which coincided with an increase in total MDH activity. A consequence of higher MDH activity was the enhanced accumulation of malate derived from dark CO2 fixation via PEPC and not from pyruvate. Although no measurable neMDH presence could be detected via PCR, gene-specific primers detected two 1kb amplicons of cMDH, designated LangMDH1 (corresponding to +P, HQ690186) and LangMDH2 (corresponding to -P, HQ690187), respectively. Sequencing analyses of these cMDH amplicons showed them to be 96% identical on an amino acid level. There was a high degree of diversification between proteins detected in this study and other known MDH proteins, particularly those from other leguminous plants. Enhanced malate synthesis in P-deficient nodules was achieved via increased anaplerotic CO2 fixation and subsequent higher MDH activities. Novel isoforms of cytosolic MDH may be involved, as shown by gene expression of specific genes under P deficiency. Copyright © 2014 Elsevier GmbH. All rights reserved.

Long Non-Coding RNA Malat1 Regulates Angiogenesis in Hindlimb Ischemia.

PubMed

Zhang, Xuejing; Tang, Xuelian; Hamblin, Milton H; Yin, Ke-Jie

2018-06-11

Angiogenesis is a complex process that depends on the delicate regulation of gene expression. Dysregulation of transcription during angiogenesis often leads to various human diseases. Emerging evidence has recently begun to show that long non-coding RNAs (lncRNAs) may mediate angiogenesis in both physiological and pathological conditions; concurrently, underlying molecular mechanisms are largely unexplored. Previously, our lab identified metastasis associates lung adenocarcinoma transcript 1 ( Malat1 ) as an oxygen-glucose deprivation (OGD)-responsive endothelial lncRNA. Here we reported that genetic deficiency of Malat1 leads to reduced blood vessel formation and local blood flow perfusion in mouse hind limbs at one to four weeks after hindlimb ischemia. Malat1 and vascular endothelial growth factor receptor 2 ( VEGFR2 ) levels were found to be increased in both cultured mouse primary skeletal muscle microvascular endothelial cells (SMMECs) after 16 h OGD followed by 24 h reperfusion and in mouse gastrocnemius muscle that underwent hindlimb ischemia followed by 28 days of reperfusion. Moreover, Malat1 silencing by locked nucleic acid (LNA)-GapmeRs significantly reduced tube formation, cell migration, and cell proliferation in SMMEC cultures. Mechanistically, RNA subcellular isolation and RNA-immunoprecipitation experiments demonstrate that Malat1 directly targets VEGFR2 to facilitate angiogenesis. The results suggest that Malat1 regulates cell-autonomous angiogenesis through direct regulation of VEGFR2.

MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple.

PubMed

Hu, Da-Gang; Sun, Cui-Hui; Sun, Mei-Hong; Hao, Yu-Jin

2016-03-01

Salt-induced phosphorylation of MdVHA-B1 protein was mediated by MdSOS2L1 protein kinase, and thereby increasing malate content in apple. Salinity is an important environmental factor that influences malate accumulation in apple. However, the molecular mechanism by which salinity regulates this process is poorly understood. In this work, we found that MdSOS2L1, a novel AtSOS2-LIKE protein kinase, interacts with V-ATPase subunit MdVHA-B1. Furthermore, MdSOS2L1 directly phosphorylates MdVHA-B1 at Ser(396) site to modulate malate accumulation in response to salt stress. Meanwhile, a series of transgenic analyses in apple calli showed that the MdSOS2L1-MdVHAB1 pathway was involved in the regulation of malate accumulation. Finally, a viral vector-based transformation approach demonstrated that the MdSOS2L1-MdVHAB1 pathway also modulated malate accumulation in apple fruits with or without salt stress. Collectively, our findings provide a new insight into the mechanism by which MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple.

Soybean NADP-Malic Enzyme Functions in Malate and Citrate Metabolism and Contributes to Their Efflux under Al Stress.

PubMed

Zhou, Ying; Yang, Zhenming; Xu, Yuezi; Sun, Haoran; Sun, Zhitao; Lin, Bao; Sun, Wenjing; You, Jiangfeng

2017-01-01

Malate accumulation has been suggested to balance Al-induced citrate synthesis and efflux in soybean roots. To test this hypothesis, characteristics of Al-induced accumulation and efflux of citrate and malate were compared between two soybean genotypes combining a functional analysis of GmME1 putatively encode a cytosolic NADP-malic enzyme. Similar amounts of citrate were released, and root elongation was equally inhibited before 8 h of Al treatment of Jiyu 70 and Jiyu 62 cultivars. Jiyu 70 began to secrete more citrate and exhibited higher Al resistance than did Jiyu 62 at 12 h. A sustained increase in internal malate and citrate concentrations was observed in Jiyu 70 at 24 h of Al treatment. However, Jiyu 62 decreased its malate concentration at 12 h and its citrate concentration at 24 h of Al treatment. GmME1 localized to the cytoplast and clustered closely with cytosolic malic enzymes AtME2 and SgME1 and was constitutively expressed in the roots. Al treatment induced higher NADP-malic enzyme activities and GmME1 expression levels in Jiyu 70 than in Jiyu 62 within 24 h. Compared with wild-type hairy roots, over-expressing GmME1 in hairy roots ( GmME1 -OE) produced higher expression levels of GmME1 but did not change the expression patterns of either of the putative citrate transporter genes GmAACT1 and GmFRDL or the malate transporter gene GmALMT1 , with or without Al treatment. GmME1 -OE showed a higher internal concentration and external efflux of both citrate and malate at 4 h of Al stress. Lighter hematoxylin staining and lower Al contents in root apices of GmME1 -OE hairy roots indicated greater Al resistance. Comprehensive experimental results suggest that sustaining Al-induced citrate efflux depends on the malate pool in soybean root apices. GmME1 encodes a cytosolic malic enzyme that contributes to increased internal malate and citrate concentrations and their external efflux to confer higher Al resistance.

Effects of chromium malate on glycometabolism, glycometabolism-related enzyme levels and lipid metabolism in type 2 diabetic rats: A dose–response and curative effects study

PubMed Central

Feng, Weiwei; Mao, Guanghua; Li, Qian; Wang, Wei; Chen, Yao; Zhao, Ting; Li, Fang; Zou, Ye; Wu, Huiyu; Yang, Liuqing; Wu, Xiangyang

2015-01-01

Aims/Introduction The present study was designed to evaluate the effect of chromium malate on glycometabolism, glycometabolism-related enzyme levels and lipid metabolism in type 2 diabetic rats, and dose–response and curative effects. Materials and Methods The model of type 2 diabetes rats was developed, and daily treatment with chromium malate was given for 4 weeks. A rat enzyme-linked immunosorbent assay kit was used to assay glycometabolism, glycometabolism-related enzyme levels and lipid metabolism changes. Results The results showed that the antihyperglycemic activity increased with administration of chromium malate in a dose–dependent manner. The serum insulin level, insulin resistance index and C-peptide level of the chromium malate groups at a dose of 17.5, 20.0 and 20.8 μg chromium/kg bodyweight were significantly lower than that of the model, chromium trichloride and chromium picolinate groups. The hepatic glycogen, glucose-6-phosphate dehydrogenase and glucokinase levels of the chromium malate groups at a dose of 17.5, 20.0 and 20.8 μg chromium/kg bodyweight were significantly higher than that of the model, chromium trichloride and chromium picolinate groups. Chromium malate at a dose of 20.0 and 20.8 μg chromium/kg bodyweight significantly changed the total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides levels compared with the chromium trichloride and chromium picolinate groups. Conclusions The results showed that chromium malate exhibits greater benefits in treating type 2 diabetes, and the curative effect of chromium malate is superior to chromium trichloride and chromium picolinate. PMID:26221518

Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weiss, P.M.; Urbauer, J.L.; Cleland, W.W.

1991-06-11

Deuterium isotope effects and {sup 13}C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the {sup 13}C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed. When dinucleotide substrates such as thio-NAD, 3-nicotinamide rings are used, the {sup 13}C effect increases when deuterated malate is the substrate comparedmore » to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the {sup 13}C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a {beta}-secondary {sup 13}C isotope effect accompanies hydride transfer as a result of hyperconjugation of the {beta}-carboxyl of malate as the transition state for the hydride transfer step is approached.« less

Succinate-driven reverse electron transport in the respiratory chain of plant mitochondria. The effects of rotenone and adenylates in relation to malate and oxaloacetate metabolism.

PubMed Central

Rustin, P; Lance, C

1991-01-01

The effects of rotenone on the succinate-driven reduction of matrix nicotinamide nucleotides were investigated in Percoll-purified mitochondria from potato (Solanum tuberosum) tubers. Depending on the presence of ADP or ATP, rotenone caused an increase or a decrease in the level of reduction of the matrix nicotinamide nucleotides. The increase in the reduction induced by rotenone in the presence of ADP was linked to the oxidation of the malate resulting from the oxidation of succinate. Depending on the experimental conditions, malic enzyme (at pH 6.6 or in the presence of added CoA) or malate dehydrogenase (at pH 7.9) were involved in this oxidation. At pH 7.9, the oxaloacetate produced progressively inhibited the succinate dehydrogenase. In the presence of ATP the production of oxaloacetate was stopped, and succinate dehydrogenase was protected from inhibition by oxaloacetate. However, previously accumulated oxaloacetate transitorily decreased the level of the reduction of the NAD+ driven by succinate, by causing the reversal of the malate dehydrogenase reaction. Under these conditions (i.e. presence of ATP), rotenone strongly inhibited the reduction of NAD+ by succinate-driven reverse electron flow. No evidence for an active reverse electron transport through a rotenone-insensitive path could be obtained. The inhibitory effect of rotenone was masked if malate had previously accumulated, owing to the malate-oxidizing enzymes which reduced part or all of the matrix NAD+. PMID:2001241

L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

PubMed

Deutch, Charles E

2013-11-01

The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

The long non-coding RNA MALAT1 promotes the migration and invasion of hepatocellular carcinoma by sponging miR-204 and releasing SIRT1.

PubMed

Hou, Zhouhua; Xu, Xuwen; Zhou, Ledu; Fu, Xiaoyu; Tao, Shuhui; Zhou, Jiebin; Tan, Deming; Liu, Shuiping

2017-07-01

Increasing evidence supports the significance of long non-coding RNA in cancer development. Several recent studies suggest the oncogenic activity of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in hepatocellular carcinoma. In this study, we explored the molecular mechanisms by which MALAT1 modulates hepatocellular carcinoma biological behaviors. We found that microRNA-204 was significantly downregulated in sh-MALAT1 HepG2 cell and 15 hepatocellular carcinoma tissues by quantitative real-time polymerase chain reaction analysis. Through bioinformatic screening, luciferase reporter assay, RNA-binding protein immunoprecipitation, and RNA pull-down assay, we identified microRNA-204 as a potential interacting partner for MALAT1. Functionally, wound-healing and transwell assays revealed that microRNA-204 significantly inhibited the migration and invasion of hepatocellular carcinoma cells. Notably, sirtuin 1 was recognized as a direct downstream target of microRNA-204 in HepG2 cells. Moreover, si-SIRT1 significantly inhibited cell invasion and migration process. These data elucidated, by sponging and competitive binding to microRNA-204, MALAT1 releases the suppression on sirtuin 1, which in turn promotes hepatocellular carcinoma migration and invasion. This study reveals a novel mechanism by which MALAT1 stimulates hepatocellular carcinoma progression and justifies targeting metastasis-associated lung adenocarcinoma transcript 1 as a potential therapy for hepatocellular carcinoma.

Malate dehydrogenase isozymes in the longnose dace, Rhinichthys cataractae.

PubMed

Starzyk, R M; Merritt, R B

1980-08-01

The interspecies homology of dace supernatant (A2,AB,B2) and mitochondrial (C2) malate dehydrogenase isozymes has been established through cell fractionation and tissue distribution studies. Isolated supernatant malate dehydrogenase (s-MDH) isozymes show significant differences in Michaelis constants for oxaloacetate and in pH optima. Shifts in s-MDH isozyme pH optima with temperature may result in immediate compensation for increase in ectotherm body pH with decrease in temperature, but duplicate s-MDH isozymes are probably maintained through selection for tissue specific regulation of metabolism.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gavva, S.R.; Harris, B.G.; Cook, P.F.

A thiol group at the malate-binding site of the NAD-malic enzyme from Ascaris suum has been modified to thiocyanate. The modified enzyme generally exhibits slight increases in K{sub NAD} and K{sub i metal} and decreases in V{sub max} as the metal size increases from Mg{sup 2+} to Mn{sup 2+} to Cd{sup 2+}, indicative of crowding in the site. The K{sub malate} value increases 10- to 30-fold, suggesting that malate does not bind optimally to the modified enzyme. Deuterium isotope effects on V and V/K{sub malate} increase with all three metal ions compared to the native enzyme concomitant with a decreasemore » in the {sup 13}C isotope effect, suggesting a switch in the rate limitation of the hydride transfer and decarboxylation steps with hydride transfer becoming more rate limiting. The {sup 13}C effect decreases only slightly when obtained with deuterated malate, suggestive of the presence of a secondary {sup 13}C effect in the hydride transfer step, similar to data obtained with non-nicotinamide-containing dinucleotide substrates for the native enzyme (see the preceding paper in this issue). The native enzyme is inactivated in a time-dependent manner by Cd{sup 2+}. This inactivation occurs whether the enzyme alone is present or whether the enzyme is turning over with Cd{sup 2+} as the divalent metal activator. Upon inactivation, only Cd{sup 2+} ions are bound at high stoichiometry to the enzyme, which eventually becomes denatured. Conversion of the active-site thiol to thiocyanate makes it more difficult to inactivate the enzyme by treatment with Cd{sup 2+}.« less

Altered Expression of the Malate-Permeable Anion Channel OsALMT4 Reduces the Growth of Rice Under Low Radiance

PubMed Central

Liu, Jie; Xu, Muyun; Estavillo, Gonzalo M.; Delhaize, Emmanuel; White, Rosemary G.; Zhou, Meixue; Ryan, Peter R.

2018-01-01

We examined the function of OsALMT4 in rice (Oryza sativa L.) which is a member of the aluminum-activated malate transporter family. Previous studies showed that OsALMT4 localizes to the plasma membrane and that expression in transgenic rice lines results in a constitutive release of malate from the roots. Here, we show that OsALMT4 is expressed widely in roots, shoots, flowers, and grain but not guard cells. Expression was also affected by ionic and osmotic stress, light and to the hormones ABA, IAA, and salicylic acid. Malate efflux from the transgenic plants over-expressing OsALMT4 was inhibited by niflumate and salicylic acid. Growth of transgenic lines with either increased OsALMT4 expression or reduced expression was measured in different environments. Light intensity caused significant differences in growth between the transgenic lines and controls. When day-time light was reduced from 700 to 300 μmol m-2s-1 independent transgenic lines with either increased or decreased OsALMT4 expression accumulated less biomass compared to their null controls. This response was not associated with differences in photosynthetic capacity, stomatal conductance or sugar concentrations in tissues. We propose that by disrupting malate fluxes across the plasma membrane carbon partitioning and perhaps signaling are affected which compromises growth under low light. We conclude that OsALMT4 is expressed widely in rice and facilitates malate efflux from different cell types. Altering OsALMT4 expression compromises growth in low-light environments. PMID:29774038

The metabolism of malate by cultured rat brain astrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

McKenna, M.C.; Tildon, J.T.; Couto, R.

1990-12-01

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-(U-14C)malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasicmore » kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain.« less

The Arabidopsis vacuolar malate channel is a member of the ALMT family.

PubMed

Kovermann, Peter; Meyer, Stefan; Hörtensteiner, Stefan; Picco, Cristiana; Scholz-Starke, Joachim; Ravera, Silvia; Lee, Youngsook; Martinoia, Enrico

2007-12-01

In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana.

PubMed

Zhao, Yannan; Luo, Lilan; Xu, Jiesi; Xin, Peiyong; Guo, Hongyan; Wu, Jian; Bai, Lin; Wang, Guodong; Chu, Jinfang; Zuo, Jianru; Yu, Hong; Huang, Xun; Li, Jiayang

2018-04-01

Programmed cell death (PCD) is a fundamental biological process. Deficiency in MOSAIC DEATH 1 (MOD1), a plastid-localized enoyl-ACP reductase, leads to the accumulation of reactive oxygen species (ROS) and PCD, which can be suppressed by mitochondrial complex I mutations, indicating a signal from chloroplasts to mitochondria. However, this signal remains to be elucidated. In this study, through cloning and analyzing a series of mod1 suppressors, we reveal a comprehensive organelle communication pathway that regulates the generation of mitochondrial ROS and triggers PCD. We show that mutations in PLASTIDIAL NAD-DEPENDENT MALATE DEHYDROGENASE (plNAD-MDH), chloroplastic DICARBOXYLATE TRANSPORTER 1 (DiT1) and MITOCHONDRIAL MALATE DEHYDROGENASE 1 (mMDH1) can each rescue the ROS accumulation and PCD phenotypes in mod1, demonstrating a direct communication from chloroplasts to mitochondria via the malate shuttle. Further studies demonstrate that these elements play critical roles in the redox homeostasis and plant growth under different photoperiod conditions. Moreover, we reveal that the ROS level and PCD are significantly increased in malate-treated HeLa cells, which can be dramatically attenuated by knockdown of the human gene MDH2, an ortholog of Arabidopsis mMDH1. These results uncover a conserved malate-induced PCD pathway in plant and animal systems, revolutionizing our understanding of the communication between organelles.

Studies on associations of glycolytic and glutaminolytic enzymes in MCF-7 cells: role of P36.

PubMed

Mazurek, S; Hugo, F; Failing, K; Eigenbrodt, E

1996-05-01

Isoelectric focusing of MCF-7 cell extracts revealed an association of the glycolytic enzymes glyceraldehyde 3-phosphate-dehydrogenase, phosphoglycerate kinase, enolase, and pyruvate kinase. This complex between the glycolytic enzymes is sensitive to RNase. p36 could not be detected within this association of glycolytic enzymes; however an association of p36 with a specific form of malate dehydrogenase was found. In MCF-7 cells three forms of malate dehydrogenase can be detected by isoelectric focusing: the mitochondrial form with an isoelectric point between 8.9 and 9.5, the cytosolic form with pl 5.0, and a p36-associated form with pl 7.8. The mitochondrial form comprises the mature mitochondrial isoenzyme (pl 9.5) and its precursor form (pl 8.9). Refocusing of the pl 7.8 form of malate dehydrogenase also gave rise to the mitochondrial isoenzyme. Thus, the pl 7.8 form of malate dehydrogenase is actually the mitochondrial isoenzyme retained in the cytosol by the association with p36. Addition of fructose 1,6-bisphosphate to the initial focusing column induced a quantitative shift of the pl 7.8 form of malate dehydrogenase to the mitochondrial forms (pl 8.9 and 9.5). In MCF-7 cells p36 is not phosphorylated in tyrosine. Kinetic measurements revealed that the pl 7.8 form of malate dehydrogenase has the lowest affinity for NADH. Compared to both mitochondrial forms the cytosolic isoenzyme has a high capacity when measured in the NAD --> NADH direction (malate --> oxaloacetate direction). The association of p36 with the mitochondrial isoenzyme may favor the flow of hydrogen from the cytosol into the mitochondria. Inhibition of cell proliferation by AMP which leads to an inhibition of glycolysis has no effect on complex formation by glycolytic and glutaminolytic enzymes in MCF-7 cells. AMP treatment leads to an activation of malate dehydrogenase, which correlates with the increase of pyruvate and the decrease of lactate levels, but has no effect on the distribution of the various malate dehydrogenase forms.

Malat1 regulates serum response factor through miR-133 as a competing endogenous RNA in myogenesis.

PubMed

Han, Xiaorui; Yang, Feng; Cao, Huiqing; Liang, Zicai

2015-07-01

Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is an example of a functional long noncoding RNA involved in many biologic processes. However, the mechanisms for Malat1 in myogenesis are unclear. Serum response factor (SRF) is a pivotal transcription factor for muscle proliferation and differentiation and is reported to be a target gene for muscle-specific microRNA-133 (miR-133). In this study, we initially found that silencing Malat1 in the mouse myoblast C2C12 cell line inhibited myocyte differentiation and decreased Srf at both the RNA and protein levels. Srf silencing decreased Malat1 expression as well. Further study revealed that Malat1 contained an miR-133 functional target site, and the interplay between Malat1 and Srf was miR-133 dependent. We demonstrated that Malat1 modulates Srf through miR-133 as a competing endogenous RNA and established a novel connection among Malat1, miR-133, and Srf in myoblast differentiation. © FASEB.

Citrulline malate enhances athletic anaerobic performance and relieves muscle soreness.

PubMed

Pérez-Guisado, Joaquín; Jakeman, Philip M

2010-05-01

The purpose of the present study was to determine the effects of a single dose of citrulline malate (CM) on the performance of flat barbell bench presses as an anaerobic exercise and in terms of decreasing muscle soreness after exercise. Forty-one men performed 2 consecutive pectoral training session protocols (16 sets). The study was performed as a randomized, double-blind, 2-period crossover design. Eight grams of CM was used in 1 of the 2 training sessions, and a placebo was used in the other. The subjects' resistance was tested using the repetitions to fatigue test, at 80% of their predetermined 1 repetition maximum (RM), in the 8 sets of flat barbell bench presses during the pectoral training session (S1-4 and S1'-4'). The p-value was 0.05. The number of repetitions showed a significant increase from placebo treatment to CM treatment from the third set evaluated (p <0.0001). This increase was positively correlated with the number of sets, achieving 52.92% more repetitions and the 100% of response in the last set (S4'). A significant decrease of 40% in muscle soreness at 24 hours and 48 hours after the pectoral training session and a higher percentage response than 90% was achieved with CM supplementation. The only side effect reported was a feeling of stomach discomfort in 14.63% of the subjects. We conclude that the use of CM might be useful to increase athletic performance in high-intensity anaerobic exercises with short rest times and to relieve postexercise muscle soreness. Thus, athletes undergoing intensive preparation involving a high level of training or in competitive events might profit from CM.

Malat1 as an evolutionarily conserved lncRNA, plays a positive role in regulating proliferation and maintaining undifferentiated status of early-stage hematopoietic cells.

PubMed

Ma, Xian-Yong; Wang, Jian-Hui; Wang, Jing-Lan; Ma, Charles X; Wang, Xiao-Chun; Liu, Feng-Song

2015-09-03

The metastasis-associated lung adenocarcinoma transcription 1 (Malat1) is a highly conserved long non-coding RNA (lncRNA) gene. Previous studies showed that Malat1 is abundantly expressed in many tissues and involves in promoting tumor growth and metastasis by modulating gene expression and target protein activities. However, little is known about the biological function and regulation mechanism of Malat1 in normal cell proliferation. In this study we conformed that Malat1 is highly conserved across vast evolutionary distances amongst 20 species of mammals in terms of sequence, and found that mouse Malat1 expresses in tissues of liver, kidney, lung, heart, testis, spleen and brain, but not in skeletal muscle. After treating erythroid myeloid lymphoid (EML) cells with All-trans Retinoic Acid (ATRA), we investigated the expression and regulation of Malat1 during hematopoietic differentiation, the results showed that ATRA significantly down regulates Malat1 expression during the differentiation of EML cells. Mouse LRH (Lin-Rhodamine(low) Hoechst(low)) cells that represent the early-stage progenitor cells show a high level of Malat1 expression, while LRB (Lin - Hoechst(Low) Rhodamine(Bright)) cells that represent the late-stage progenitor cells had no detectable expression of Malat1. Knockdown experiment showed that depletion of Malat1 inhibits the EML cell proliferation. Along with the down regulation of Malat1, the tumor suppressor gene p53 was up regulated during the differentiation. Interestingly, we found two p53 binding motifs with help of bioinformatic tools, and the following chromatin immunoprecipitation (ChIP) test conformed that p53 acts as a transcription repressor that binds to Malat1's promoter. Furthermore, we testified that p53 over expression in EML cells causes down regulation of Malat1. In summary, this study indicates Malat1 plays a critical role in maintaining the proliferation potential of early-stage hematopoietic cells. In addition to its biological function, the study also uncovers the regulation pattern of Malat1 expression mediated by p53 in hematopoietic differentiation. Our research shed a light on exploring the Malat1 biological role including therapeutic significance to inhibit the proliferation potential of malignant cells.

A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase

PubMed Central

Irague, Romain; Topham, Christopher M.; Martineau, Nelly; Baylac, Audrey; Auriol, Clément; Walther, Thomas; François, Jean-Marie; Remaud-Siméon, Magali

2018-01-01

An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (kcat/Km)malate/(kcat/Km)aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site. PMID:29462203

Physical Fitness and Mitochondrial Respiratory Capacity in Horse Skeletal Muscle

PubMed Central

Lemieux, Hélène; Mouithys-Mickalad, Ange; Serteyn, Didier

2012-01-01

Background Within the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general. Methodology/Principal Findings We applied high-resolution respirometry and multiple substrate-uncoupler-inhibitor titration protocols to study mitochondrial physiology in small (1.0–2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses. Oxidative phosphorylation (OXPHOS) capacity (pmol O2•s−1•mg−1 wet weight) with combined Complex I and II (CI+II) substrate supply (malate+glutamate+succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained and competitive horses (N = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 (N = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the CI+II substrate cocktail. Relative to this maximum OXPHOS capacity, Complex I (CI)-linked OXPHOS capacities were only 50% with glutamate+malate, 64% with pyruvate+malate, and 68% with pyruvate+malate+glutamate, and ∼78% with CII-linked succinate+rotenone. OXPHOS capacity with glutamate+malate increased with fitness relative to CI+II-supported ETS capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the CII/CI+II substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the CI- over CII-linked pathway capacity was reduced with physical fitness. Conclusions/Significance The scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of CI-linked respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology. PMID:22529950

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.

PubMed

Zelle, Rintze M; de Hulster, Erik; van Winden, Wouter A; de Waard, Pieter; Dijkema, Cor; Winkler, Aaron A; Geertman, Jan-Maarten A; van Dijken, Johannes P; Pronk, Jack T; van Maris, Antonius J A

2008-05-01

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

Relayed 13C magnetization transfer: Detection of malate dehydrogenase reaction in vivo

NASA Astrophysics Data System (ADS)

Yang, Jehoon; Shen, Jun

2007-02-01

Malate dehydrogenase catalyzes rapid interconversion between dilute metabolites oxaloacetate and malate. Both oxaloacetate and malate are below the detection threshold of in vivo MRS. Oxaloacetate is also in rapid exchange with aspartate catalyzed by aspartate aminotransferase, the latter metabolite is observable in vivo using 13C MRS. We hypothesized that the rapid turnover of oxaloacetate can effectively relay perturbation of magnetization between malate and aspartate. Here, we report indirect observation of the malate dehydrogenase reaction by saturating malate C2 resonance at 71.2 ppm and detecting a reduced aspartate C2 signal at 53.2 ppm due to relayed magnetization transfer via oxaloacetate C2 at 201.3 ppm. Using this strategy the rate of the cerebral malate dehydrogenase reaction was determined to be 9 ± 2 μmol/g wet weight/min (means ± SD, n = 5) at 11.7 Tesla in anesthetized adult rats infused with [1,6- 13C 2]glucose.

Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism.

PubMed

Ralphe, J Carter; Bedell, Kurt; Segar, Jeffrey L; Scholz, Thomas D

2005-05-01

In the heart, elevated thyroid hormone leads to upregulation of metabolic pathways associated with energy production and development of hypertrophy. The malate/aspartate shuttle, which transfers cytosolic-reducing equivalents into the cardiac mitochondria, is increased 33% in hyperthyroid rats. Within the shuttle, the aspartate-glutamate carrier is rate limiting. The excitatory amino acid transporter type 1 (EAAT1) functions as a glutamate carrier in the malate/aspartate shuttle. In this study, we hypothesize that EAAT1 is regulated by thyroid hormone. Adult rats were injected with triiodothyronine (T3) or saline over a period of 8-9 days or provided with propylthiouracil (PTU) in their drinking water for 2 mo. Steady-state mRNA levels of EAAT1 and aralar1 and citrin (both cardiac mitochondrial aspartate-glutamate transporters) were determined by Northern blot analysis and normalized to 18S rRNA. A spectrophotometric assay of maximal malate/aspartate shuttle activity was performed on isolated cardiac mitochondria from PTU-treated and control animals. Protein lysates from mitochondria were separated by SDS-PAGE and probed with a human anti-EAAT1 IgG. Compared with control, EAAT1 mRNA levels (arbitrary units) were increased nearly threefold in T3-treated (3.1 +/- 0.5 vs. 1.1 +/- 0.2; P < 0.05) and decreased in PTU-treated (2.0 +/- 0. 3 vs. 5.2 +/- 1; P < 0.05) rats. Aralar1 mRNA levels were unchanged in T3-treated and somewhat decreased in PTU-treated (7.1 +/- 1.0 vs. 9.3 +/- 0.1, P < 0.05) rats. Citrin mRNA levels were decreased in T3-treated and unchanged in PTU-treated rats. EAAT1 protein levels (arbitrary units) in T3-treated cardiac mitochondria were increased compared with controls (8.9 +/- 0.4 vs. 5.9 +/- 0.6; P < 0.005) and unchanged in PTU-treated mitochondria. No difference in malate/aspartate shuttle capacity was found between PTU-treated and control cardiac mitochondria. Hyperthyroidism in rats is related to an increase in cardiac expression of EAAT1 mRNA and protein. The 49% increase in EAAT1 mitochondrial protein level shows that malate/aspartate shuttle activity increased in hyperthyroid rat cardiac mitochondria. Although hypothyroidism resulted in a decrease in EAAT1 mRNA, neither the EAAT1 protein level nor shuttle activity was affected. EAAT1 regulation by thyroid hormone may facilitate increased metabolic demands of the cardiomyocyte during hyperthyroidism and impact cardiac function in hyperthyroidism.

Hypoxia-induced long non-coding RNA Malat1 is dispensable for renal ischemia/reperfusion-injury.

PubMed

Kölling, Malte; Genschel, Celina; Kaucsar, Tamas; Hübner, Anika; Rong, Song; Schmitt, Roland; Sörensen-Zender, Inga; Haddad, George; Kistler, Andreas; Seeger, Harald; Kielstein, Jan T; Fliser, Danilo; Haller, Hermann; Wüthrich, Rudolf; Zörnig, Martin; Thum, Thomas; Lorenzen, Johan

2018-02-21

Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury (AKI). Non-coding RNAs are crucially involved in its pathophysiology. We identified hypoxia-induced long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) to be upregulated in renal I/R injury. We here elucidated the functional role of Malat1 in vitro and its potential contribution to kidney injury in vivo. Malat1 was upregulated in kidney biopsies and plasma of patients with AKI, in murine hypoxic kidney tissue as well as in cultured and ex vivo sorted hypoxic endothelial cells and tubular epithelial cells. Malat1 was transcriptionally activated by hypoxia-inducible factor 1-α. In vitro, Malat1 inhibition reduced proliferation and the number of endothelial cells in the S-phase of the cell cycle. In vivo, Malat1 knockout and wildtype mice showed similar degrees of outer medullary tubular epithelial injury, proliferation, capillary rarefaction, inflammation and fibrosis, survival and kidney function. Small-RNA sequencing and whole genome expression analysis revealed only minor changes between ischemic Malat1 knockout and wildtype mice. Contrary to previous studies, which suggested a prominent role of Malat1 in the induction of disease, we did not confirm an in vivo role of Malat1 concerning renal I/R-injury.

The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier

PubMed Central

Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A.; Traub, Michaela; Martinoia, Enrico; Neuhaus, H. Ekkehard

2003-01-01

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter. PMID:12947042

The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier.

PubMed

Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A; Traub, Michaela; Martinoia, Enrico; Neuhaus, H Ekkehard

2003-09-16

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter.

Metabolism of pyruvate and malate by isolated fat-cell mitochondria.

PubMed

Martin, B R; Denton, R M

1971-11-01

1. Metabolism of pyruvate and malate by isolated fat-cell mitochondria incubated in the presence of ADP and phosphate has been studied by measuring rates of pyruvate uptake, malate utilization or production, citrate production and oxygen consumption. From these measurements calculations of the flow rates through pyruvate carboxylase, pyruvate dehydrogenase and citrate cycle have been made under various conditions. 2. In the presence of bicarbonate, pyruvate was largely converted into citrate and malate and only about 10% was oxidized by the citrate cycle; citrate and malate outputs were linear after lag periods of 6-9min and 3min respectively, and no other end products of pyruvate metabolism were detected. On the further addition of malate or hydroxymalonate, the lag in the rate of citrate output was less marked but no net malate disappearance was detected. If, however, bicarbonate was omitted then net malate uptake was observed. Addition of butyl malonate was found to greatly inhibit the metabolism of pyruvate to citrate and malate in the presence of bicarbonate. 3. These results are in agreement with earlier conclusions that in adipose tissue acetyl units for fatty acid synthesis are transferred to the cytoplasm as citrate and that this transfer requires malate presumably for counter transport. They also support the view that oxaloacetate for citrate synthesis is preferentially formed from pyruvate through pyruvate carboxylase rather than malate through malate dehydrogenase and that the mitochondrial metabolism of citrate in fat-cells is restricted. The possible consequences of these conclusions are discussed. 4. Studies on the effects of additions of adenine nucleotides to pyruvate metabolism by isolated fat-cell mitochondria are consistent with inhibition of pyruvate carboxylase in the presence of ADP and pyruvate dehydrogenase in the presence of ATP.

Down-regulation of Long Noncoding RNA MALAT1 Protects Hippocampal Neurons Against Excessive Autophagy and Apoptosis via the PI3K/Akt Signaling Pathway in Rats with Epilepsy.

PubMed

Wu, Qiang; Yi, Xuewei

2018-06-01

Epilepsy is a common chronic brain disorder and is characterized by an enduring predisposition to generate seizures. The hippocampus is especially vulnerable to seizure-induced damage. In this study, we explore the ability of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) to influence the autophagy and apoptosis of hippocampal neurons in epilepsy and the underlying mechanism involving the PI3K/Akt signaling pathway. Seventy-two Sprague-Dawley rats were assigned to normal, sham, Ep, Ep + si-NC, Ep + si-MALAT1, and Ep + si-MALAT1 + LY groups. Fluorescence in situ hybridization kit was employed to determine the MALAT1 in the brain tissues. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were performed to determine the expression of MALAT1, mRNAs, and proteins. The autophagy of hippocampal neurons was evaluated under a transmission electron microscope and their apoptosis was evaluated using TUNEL staining. We found that MALAT1 and c-Met were enriched while microRNA-101 (miR-101) decreased in rats with epilepsy. The demonstration showed that MALAT1 binds to miR-101, thus regulating c-Met. In rats with epilepsy, MALAT1 depletion mediated by anti-MALAT1 siRNA resulted in activation of PI3K/Akt signaling pathway and loss of hippocampal neurons. LY294002, an inhibitor of PI3K/Akt signaling pathway, could reverse the events caused by MALAT1 knockdown. Taken together, these findings indicate that down-regulation of MALAT1 activates the PI3K/Akt signaling pathway to protect hippocampal neurons against autophagy and apoptosis in rats with epilepsy.

Association of twelve polymorphisms in three onco-lncRNA genes with hepatocellular cancer risk and prognosis: A case-control study

PubMed Central

Wang, Ben-Gang; Xu, Qian; Lv, Zhi; Fang, Xin-Xin; Ding, Han-Xi; Wen, Jing; Yuan, Yuan

2018-01-01

AIM To evaluate the association of 12 tag single nucleotide polymorphisms (tagSNPs) in three onco-long non-coding RNA (lncRNA) genes (HOTTIP, CCAT2, MALAT1) with the risk and prognosis of hepatocellular cancer (HCC). METHODS Twelve tagSNPs covering the three onco-lncRNAs were genotyped by the KASP method in a total of 1338 samples, including 521 HCC patients and frequency-matched 817 controls. The samples were obtained from an unrelated Chinese population at the First Hospital of China Medical University from 2012-2015. The expression quantitative trait loci (eQTL) analyses were conducted to explore further the potential function of the promising SNPs. RESULTS Three SNPs in HOTTIP, one promoter SNP in MALAT1, and one haplotype of HOTTIP were associated with HCC risk. The HOTTIP rs17501292, rs2067087, and rs17427960 SNPs were increased to 1.55-, 1.20-, and 1.18-fold HCC risk under allelic models (P = 0.012, 0.017 and 0.049, respectively). MALAT1 rs4102217 SNP was increased to a 1.32-fold HCC risk under dominant models (P = 0.028). In addition, the two-way interaction of HOTTIP rs17501292-MALAT1 rs619586 polymorphisms showed a decreased effect on HCC risk (Pinteraction = 0.028, OR = 0.30) and epistasis with each other. HOTTIP rs3807598 variant genotype showed significantly longer survival time in HBV negative subgroup (P = 0.049, HR = 0.12), and MALAT1 rs591291 showed significantly better prognosis in female and HBV negative subgroups (P = 0.022, HR = 0.37; P = 0.042, HR = 0.25, respectively). In the study, no significant effect was observed in eQTL analysis. CONCLUSION Specific lncRNA (HOTTIP and MALAT1) SNPs have potential to be biomarkers for HCC risk and prognosis. PMID:29930469

Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury

PubMed Central

WEINBERG, JOEL M.; VENKATACHALAM, MANJERI A.; ROESER, NANCY F.; SAIKUMAR, POTHANA; DONG, ZHENG; SENTER, RUTH A.; NISSIM, ITZHAK

2010-01-01

We have further examined the mechanisms for a severe mitochondrial energetic deficit, deenergization, and impaired respiration in complex I that develop in kidney proximal tubules during hypoxia-reoxygenation, and their prevention and reversal by supplementation with α-ketoglutarate (α-KG) + aspartate. The abnormalities preceded the mitochondrial permeability transition and cytochrome c loss. Anaerobic metabolism of α-KG + aspartate generated ATP and maintained mitochondrial membrane potential. Other citric-acid cycle intermediates that can promote anaerobic metabolism (malate and fumarate) were also effective singly or in combination with α-KG. Succinate, the end product of these anaerobic pathways that can bypass complex I, was not protective when provided only during hypoxia. However, during reoxygenation, succinate also rescued the tubules, and its benefit, like that of α-KG + malate, persisted after the extra substrate was withdrawn. Thus proximal tubules can be salvaged from hypoxia-reoxygenation mitochondrial injury by both anaerobic metabolism of citric-acid cycle intermediates and aerobic metabolism of succinate. These results bear on the understanding of a fundamental mode of mitochondrial dysfunction during tubule injury and on strategies to prevent and reverse it. PMID:11053054

Glutamine, glutamate, and other possible regulators of alpha-ketoglutarate and malate uptake by synaptic terminals.

PubMed

Shank, R P; Campbell, G L

1984-04-01

The uptake of alpha-ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of alpha-ketoglutarate by causing an apparent reduction in the substrate-carrier affinity and an increase in Vmax. In contrast, glutamine did not appear to affect the Vmax of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L-Glutamate and L-aspartate were comparatively strong inhibitors of alpha-ketoglutarate and malate uptake. N-Acetylaspartate was a weak inhibitor of alpha-ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited alpha-ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8- to 14-day-old mice. Ca2+ exhibited a variable effect but usually enhanced the uptake of alpha-ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of alpha-ketoglutarate by low-density synaptosomes. By comparison, the uptake of glutamate, glutamine, gamma-aminobutyric acid, and several other amino acids was not affected. The enhancement of alpha-ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U-14C] alpha-ketoglutarate to glutamate, aspartate, glutamine, and gamma-aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably.

Modeling the vacuolar storage of malate shed lights on pre- and post-harvest fruit acidity.

PubMed

Etienne, Audrey; Génard, Michel; Lobit, Philippe; Bugaud, Christophe

2014-11-18

Malate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. Several studies suggest that malate accumulation in fruit cells is controlled at the level of vacuolar storage. However, the regulation of vacuolar malate storage throughout fruit development, and the origins of the phenotypic variability of the malate concentration within fruit species remain to be clarified. In the present study, we adapted the mechanistic model of vacuolar storage proposed by Lobit et al. in order to study the accumulation of malate in pre and postharvest fruits. The main adaptation concerned the variation of the free energy of ATP hydrolysis during fruit development. Banana fruit was taken as a reference because it has the particularity of having separate growth and post-harvest ripening stages, during which malate concentration undergoes substantial changes. Moreover, the concentration of malate in banana pulp varies greatly among cultivars which make possible to use the model as a tool to analyze the genotypic variability. The model was calibrated and validated using data sets from three cultivars with contrasting malate accumulation, grown under different fruit loads and potassium supplies, and harvested at different stages. The model predicted the pre and post-harvest dynamics of malate concentration with fairly good accuracy for the three cultivars (mean RRMSE = 0.25-0.42). The sensitivity of the model to parameters and input variables was analyzed. According to the model, vacuolar composition, in particular potassium and organic acid concentrations, had an important effect on malate accumulation. The model suggested that rising temperatures depressed malate accumulation. The model also helped distinguish differences in malate concentration among the three cultivars and between the pre and post-harvest stages by highlighting the probable importance of proton pump activity and particularly of the free energy of ATP hydrolysis and vacuolar pH. This model appears to be an interesting tool to study malate accumulation in pre and postharvest fruits and to get insights into the ecophysiological determinants of fruit acidity, and thus may be useful for fruit quality improvement.

Long noncoding RNA MALAT1 as a potential novel biomarker in digestive system cancers: a meta-analysis.

PubMed

Song, Wei; Zhang, Run J; Zou, Shu B

2016-08-01

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a newly discovered long non-coding RNA (lncRNA), has been reported to be overexpressed in various cancers. However, the clinical value of MALAT1 in digestive system cancers is unclear. This study was designed to investigate the potential value of MALAT1 as a prognostic biomarker in digestive system cancers. We searched the Medline, Embase and Cochrane Library databases. All studies that explored the correlation between lncRNA MALAT1 expression and survival in digestive system tumors were selected. A quantitative meta-analysis was performed for the correlation between lncRNA MALAT1 expression and survival in digestive system tumors. Five studies were eligible for analysis, which included 547 patients. Meta-analysis showed that high expression of MALAT1 could predict poor overall survival (OS) in digestive system cancers (pooled HR: 1.85, 95% CI: 1.41-2.43, P<0.0001). For disease-free survival (DFS), elevated MALAT1 expression was also a significant predictor with a combined HR of 2.28 (95% CI: 1.42-3.67, P=0.0007). lncRNA MALAT1 may serve as a potential novel prognostic biomarker in digestive system cancers.

Long noncoding RNA MALAT1 as a potential novel biomarker in digestive system cancers: a meta-analysis.

PubMed

Song, Wei; Zhang, Run J; Zou, Shu B

2016-05-17

MALAT1 (Metastasis-associated lung adenocarcinoma transcript 1), a newly discovered long non-coding RNA (lncRNA), has been reported to be overexpressed in various cancers. However, the clinical value of MALAT1 in digestive system cancers is unclear. This study was designed to investigate the potential value of MALAT1 as a prognostic biomarker in digestive system cancers. We searched the MEDLINE, EMBASE and Cochrane Library databases. All studies that explored the correlation between lncRNA MALAT1 expression and survival in digestive system tumors were selected. A quantitative meta-analysis was performed for the correlation between lncRNA MALAT1 expression and survival in digestive system tumors. Five studies were eligible for analysis, which included 547 patients. Meta-analysis showed that high expression of MALAT1 could predict poor overall survival (OS) in digestive system cancers (pooled HR: 1.85, 95% CI: 1.41-2.43, p < 0.0001). For disease-free survival (DFS), elevated MALAT1 expression was also a significant predictor with a combined HR of 2.28 (95% CI: 1.42-3.67, p = 0.0007). lncRNA MALAT1 may serve as a potential novel prognostic biomarker in digestive system cancers.

The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.

PubMed

De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

2013-08-01

Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA.

Evidence That Isoprene Emission Is Not Limited by Cytosolic Metabolites. Exogenous Malate Does Not Invert the Reverse Sensitivity of Isoprene Emission to High [CO2].

PubMed

Rasulov, Bahtijor; Talts, Eero; Bichele, Irina; Niinemets, Ülo

2018-02-01

Isoprene is synthesized via the chloroplastic 2- C -methyl-d-erythritol 4-phosphate/1-deoxy-d-xylulose 5-phosphate pathway (MEP/DOXP), and its synthesis is directly related to photosynthesis, except under high CO 2 concentration, when the rate of photosynthesis increases but isoprene emission decreases. Suppression of MEP/DOXP pathway activity by high CO 2 has been explained either by limited supply of the cytosolic substrate precursor, phospho enol pyruvate (PEP), into chloroplast as the result of enhanced activity of cytosolic PEP carboxylase or by limited supply of energetic and reductive equivalents. We tested the PEP-limitation hypotheses by feeding leaves with the PEP carboxylase competitive inhibitors malate and diethyl oxalacetate (DOA) in the strong isoprene emitter hybrid aspen ( Populus tremula × Populus tremuloides ). Malate feeding resulted in the inhibition of net assimilation, photosynthetic electron transport, and isoprene emission rates, but DOA feeding did not affect any of these processes except at very high application concentrations. Both malate and DOA did not alter the sensitivity of isoprene emission to high CO 2 concentration. Malate inhibition of isoprene emission was associated with enhanced chloroplastic reductive status that suppressed light reactions of photosynthesis, ultimately leading to reduced isoprene substrate dimethylallyl diphosphate pool size. Additional experiments with altered oxygen concentrations in conditions of feedback-limited and non-feedback-limited photosynthesis further indicated that changes in isoprene emission rate in control and malate-inhibited leaves were associated with changes in the share of ATP and reductive equivalent supply for isoprene synthesis. The results of this study collectively indicate that malate importantly controls the chloroplast reductive status and, thereby, affects isoprene emission, but they do not support the hypothesis that cytosolic metabolite availability alters the response of isoprene emission to changes in atmospheric composition. © 2018 American Society of Plant Biologists. All Rights Reserved.

Long Non-Coding RNA MALAT1 Interacts With miR-204 to Modulate Human Hilar Cholangiocarcinoma Proliferation, Migration, and Invasion by Targeting CXCR4.

PubMed

Tan, Xinyu; Huang, Zhiguo; Li, Xiaogang

2017-11-01

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is involved in the development and progression of many types of tumors. An aberrant expression of MALAT1 was observed in many kinds of cancers. However, the exact effects and molecular mechanisms of MALAT1 in human hilar cholangiocarcinoma (HCCA) progression are still unknown. Here, we investigated the role of MALAT1 in human HCCA cell lines and clinical tumor samples in order to determine the function of this lncRNA. In our research, lncRNA-MALAT1 was specifically upregulated in HCCA tissues and cell lines, and was associated with pathological T stage, a larger tumor size, and perineural invasion. Knockdown of MALAT1 inhibited the proliferation, migration, and invasion of human HCCA cell. In addition, chemokine receptor-4 (CXCR4) was involved in MALAT1 induced human HCCA growth, migration, and invasion. By using online tools and a series of mechanistic analysis, we also demonstrated that miR-204-dependent CXCR4 regulation was required in MALAT1 modulating HCCA cell growth, migration and invasion. Taken together, our data indicated that MALAT1 might play an oncogenic role in HCCA through miR-204-dependent CXCR4 regulation, and could be regarded as a therapeutic target in HCCA. J. Cell. Biochem. 118: 3643-3653, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

LncRNA MALAT1 sponges miR-204 to promote osteoblast differentiation of human aortic valve interstitial cells through up-regulating Smad4.

PubMed

Xiao, Xiaoxiong; Zhou, Tingwen; Guo, Shichao; Guo, Chao; Zhang, Qiao; Dong, Nianguo; Wang, Yongjun

2017-09-15

Emerging evidences have indicated that long non-coding RNAs (lncRNAs) play vital roles in cardiovascular physiology and pathology. The lncRNA MALAT1, a highly abundant and conserved imprinted gene, has been implicated in many cardiovascular diseases. However, the function of MALAT1 in calcific aortic valve disease (CAVD) remains unknown. This study sought to document the function and underlying mechanism of MALAT1 in regulating CAVD. Protein level was determined by immunoblotting and immunofluorescence staining. MALAT1, miR-204 and mRNA expressions were detected by qRT-PCR. Mineralized bone matrix formation was assessed by Alizarin Red staining. The interaction between MALAT1 and miR-204 was studied using luciferase reporter assay, RNA pull-down assay and RNA-binding protein immunoprecipitation assay. Ectopic expression of MALAT1 was observed in calcific valves and after osteogenic induction in human aortic valve interstitial cells (VICs). In vitro experiments revealed that MALAT1 acted as a positive regulator of osteogenic differentiation by repressing miR-204 expression and activity and thereby promoting expression of osteoblast-specific markers, including alkaline phosphatase, mineralized bone matrix formation and osteocalcin. Mechanistically, we identified Smad4 as a direct target of miR-204. Importantly, MALAT1 could directly interact with miR-204 and overexpression of miR-204 efficiently reversed the upregulation of Smad4 induced by MALAT1. Thus, MALAT1 positively regulated the expression of Smad4 through sponging miR-204, and promoted osteogenic differentiation of VICs. Our study provides novel mechanistic insights into a critical role for lncRNA MALAT1 as a miRNA sponge in CAVD and sheds new light on lncRNA-directed diagnostics and therapeutics in CAVD. Copyright © 2017. Published by Elsevier B.V.

An InDel in the promoter of Al-activated malate transporter 9 selected during tomato domestication determines fruit malate content and aluminum tolerance

USDA-ARS?s Scientific Manuscript database

Deciphering the mechanism of malate accumulation in plants would contribute to a greater understanding of plant chemistry, which has implications for improving flavor quality in crop species and enhancing human health benefits. However, the regulation of malate metabolism is poorly understood in cro...

Impaired Malate and Fumarate Accumulation Due to the Mutation of the Tonoplast Dicarboxylate Transporter Has Little Effects on Stomatal Behavior.

PubMed

Medeiros, David B; Barros, Kallyne A; Barros, Jessica Aline S; Omena-Garcia, Rebeca P; Arrivault, Stéphanie; Sanglard, Lílian M V P; Detmann, Kelly C; Silva, Willian Batista; Daloso, Danilo M; DaMatta, Fábio M; Nunes-Nesi, Adriano; Fernie, Alisdair R; Araújo, Wagner L

2017-11-01

Malate is a central metabolite involved in a multiplicity of plant metabolic pathways, being associated with mitochondrial metabolism and playing significant roles in stomatal movements. Vacuolar malate transport has been characterized at the molecular level and is performed by at least one carrier protein and two channels in Arabidopsis ( Arabidopsis thaliana ) vacuoles. The absence of the Arabidopsis tonoplast Dicarboxylate Transporter (tDT) in the tdt knockout mutant was associated previously with an impaired accumulation of malate and fumarate in leaves. Here, we investigated the consequences of this lower accumulation on stomatal behavior and photosynthetic capacity as well as its putative metabolic impacts. Neither the stomatal conductance nor the kinetic responses to dark, light, or high CO 2 were highly affected in tdt plants. In addition, we did not observe any impact on stomatal aperture following incubation with abscisic acid, malate, or citrate. Furthermore, an effect on photosynthetic capacity was not observed in the mutant lines. However, leaf mitochondrial metabolism was affected in the tdt plants. Levels of the intermediates of the tricarboxylic acid cycle were altered, and increases in both light and dark respiration were observed. We conclude that manipulation of the tonoplastic organic acid transporter impacted mitochondrial metabolism, while the overall stomatal and photosynthetic capacity were unaffected. © 2017 American Society of Plant Biologists. All Rights Reserved.

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

PubMed Central

Hebbelmann, Inga; Selinski, Jennifer; Wehmeyer, Corinna; Goss, Tatjana; Voss, Ingo; Mulo, Paula; Kangasjärvi, Saijaliisa; Aro, Eva-Mari; Oelze, Marie-Luise; Dietz, Karl-Josef; Nunes-Nesi, Adriano; Do, Phuc T.; Fernie, Alisdair R.; Talla, Sai K.; Raghavendra, Agepati S.; Linke, Vera; Scheibe, Renate

2012-01-01

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants. PMID:22140244

Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

PubMed

Newsom, Sean A; Miller, Benjamin F; Hamilton, Karyn L; Ehrlicher, Sarah E; Stierwalt, Harrison D; Robinson, Matthew M

2017-11-01

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H 2 O 2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance. Copyright © 2017 the American Physiological Society.

Long non-coding RNA metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) interacts with estrogen receptor and predicted poor survival in breast cancer.

PubMed

Huang, Nai-Si; Chi, Ya-Yun; Xue, Jing-Yan; Liu, Meng-Ying; Huang, Sheng; Mo, Miao; Zhou, Shu-Ling; Wu, Jiong

2016-06-21

Metastasis associated in lung adenocarcinoma transcript 1 (MALAT1), a lncRNA that was first recognized as a prognostic parameter for patient survival of stage I lung cancer, is up-regulated in multiple human malignancies, including breast cancer. However, the mechanism of its function remained elusive. In the current study, by examining MALAT1 expression on mRNA level, we demonstrated that compared with MCF10A, MALAT1 expression was up-regulated in the majority of breast cancer cell lines (9/12). In 26 pairs of estrogen receptor (ER)-positive breast cancer samples, MALAT1 expression was significantly up-regulated compared with adjacent normal tissues (P = 0.012). Furthermore, of 204 breast cancer patients, high MALAT1 expression was associated with positive ER (P = 0.023) and progesterone receptor (PR) (P = 0.024) status. Further analysis using TCGA database revealed that ER and its target genes PGR and CCND1, were overexpressed in MALAT1 altered group compared with unaltered group, both on the mRNA and protein level. Lastly, we verified MALAT1's prognostic value in breast cancer. At the cut-off value of 75%, MALAT1 was the only independent prognostic factor of recurrence-free survival (RFS) in ER-negative patients in a multivariate Cox regression model (hazard ratio [HR] = 2.83, 95% confidence interval [CI] 1.02-7.83). MALAT1 overexpression was also associated with poor RFS in tamoxifen treated ER-positive breast cancer patients, which might serve as a potential biomarker to predict endocrine treatment sensitivity.

Increased excitability and metabolism in pilocarpine induced epileptic rats: effect of Bacopa monnieri.

PubMed

Mathew, Jobin; Paul, Jes; Nandhu, M S; Paulose, C S

2010-09-01

We have evaluated the acetylcholine esterase and malate dehydrogenase activity in the muscle, epinephrine, norepinephrine, insulin and T3 content in the serum of epileptic rats. Acetylcholine esterase and malate dehydrogenase activity increased in the muscle and decreased in the heart of the epileptic rats compared to control. Insulin and T3 content were increased significantly in the serum of the epileptic rats. Our results suggest that repetitive seizures resulted in increased metabolism and excitability in epileptic rats. Bacopa monnieri and Bacoside-A treatment prevents the occurrence of seizures there by reducing the impairment on peripheral nervous system. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit.

PubMed

Sweetman, C; Sadras, V O; Hancock, R D; Soole, K L; Ford, C M

2014-11-01

Berries of the cultivated grapevine Vitis vinifera are notably responsive to temperature, which can influence fruit quality and hence the future compatibility of varieties with their current growing regions. Organic acids represent a key component of fruit organoleptic quality and their content is significantly influenced by temperature. The objectives of this study were to (i) manipulate thermal regimes to realistically capture warming-driven reduction of malate content in Shiraz berries, and (ii) investigate the mechanisms behind temperature-sensitive malate loss and the potential downstream effects on berry metabolism. In the field we compared untreated controls at ambient temperature with longer and milder warming (2-4 °C differential for three weeks; Experiment 1) or shorter and more severe warming (4-6 °C differential for 11 days; Experiment 2). We complemented field trials with control (25/15 °C) and elevated (35/20 °C) day/night temperature controlled-environment trials using potted vines (Experiment 3). Elevating maximum temperatures (4-10 °C above controls) during pre-véraison stages led to higher malate content, particularly with warmer nights. Heating at véraison and ripening stages reduced malate content, consistent with effects typically seen in warm vintages. However, when minimum temperatures were also raised by 4-6 °C, malate content was not reduced, suggesting that the regulation of malate metabolism differs during the day and night. Increased NAD-dependent malic enzyme activity and decreased phosphoenolpyruvate carboxylase and pyruvate kinase activities, as well as the accumulation of various amino acids and γ-aminobutyric acid, suggest enhanced anaplerotic capacity of the TCA cycle and a need for coping with decreased cytosolic pH in heated fruit. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit

PubMed Central

Sweetman, C.; Sadras, V. O.; Hancock, R. D.; Soole, K. L.; Ford, C. M.

2014-01-01

Berries of the cultivated grapevine Vitis vinifera are notably responsive to temperature, which can influence fruit quality and hence the future compatibility of varieties with their current growing regions. Organic acids represent a key component of fruit organoleptic quality and their content is significantly influenced by temperature. The objectives of this study were to (i) manipulate thermal regimes to realistically capture warming-driven reduction of malate content in Shiraz berries, and (ii) investigate the mechanisms behind temperature-sensitive malate loss and the potential downstream effects on berry metabolism. In the field we compared untreated controls at ambient temperature with longer and milder warming (2–4 °C differential for three weeks; Experiment 1) or shorter and more severe warming (4–6 °C differential for 11 days; Experiment 2). We complemented field trials with control (25/15 °C) and elevated (35/20 °C) day/night temperature controlled-environment trials using potted vines (Experiment 3). Elevating maximum temperatures (4–10 °C above controls) during pre-véraison stages led to higher malate content, particularly with warmer nights. Heating at véraison and ripening stages reduced malate content, consistent with effects typically seen in warm vintages. However, when minimum temperatures were also raised by 4–6 °C, malate content was not reduced, suggesting that the regulation of malate metabolism differs during the day and night. Increased NAD-dependent malic enzyme activity and decreased phosphoenolpyruvate carboxylase and pyruvate kinase activities, as well as the accumulation of various amino acids and γ-aminobutyric acid, suggest enhanced anaplerotic capacity of the TCA cycle and a need for coping with decreased cytosolic pH in heated fruit. PMID:25180109

Action of diclofenac on kidney mitochondria and cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ng, Lin Eng; Vincent, Annette S.; Halliwell, Barry

2006-09-22

The mitochondrial membrane potential measured in isolated rat kidney mitochondria and in digitonin-permeabilized MDCK type II cells pre-energized with succinate, glutamate, and/or malate was reduced by micromolar diclofenac dose-dependently. However, ATP biosynthesis from glutamate/malate was significantly more compromised compared to that from succinate. Inhibition of the malate-aspartate shuttle by diclofenac with a resultant decrease in the ability of mitochondria to generate NAD(P)H was demonstrated. Diclofenac however had no effect on the activities of NADH dehydrogenase, glutamate dehydrogenase, and malate dehydrogenase. In conclusion, decreased NAD(P)H production due to an inhibition of the entry of malate and glutamate via the malate-aspartate shuttlemore » explained the more pronounced decreased rate of ATP biosynthesis from glutamate and malate by diclofenac. This drug, therefore affects the bioavailability of two major respiratory complex I substrates which would normally contribute substantially to supplying the reducing equivalents for mitochondrial electron transport for generation of ATP in the renal cell.« less

Long non-coding RNA MALAT1 acts as a competing endogenous RNA to promote malignant melanoma growth and metastasis by sponging miR-22.

PubMed

Luan, Wenkang; Li, Lubo; Shi, Yan; Bu, Xuefeng; Xia, Yun; Wang, Jinlong; Djangmah, Henry Siaw; Liu, Xiaohui; You, Yongping; Xu, Bin

2016-09-27

Long non-coding RNAs (lncRNAs) are involved in tumorigenesis. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an lncRNAs, is associated with the growth and metastasis of many human tumors, but its biological roles in malignant melanoma remain unclear. In this study, the aberrant up-regulation of MALAT1 was detected in melanoma. We determined that MALAT1 promotes melanoma cells proliferation, invasion and migration by sponging miR-22. MiR-22 was decreased and acted as a tumor suppressor in melanoma, and MMP14 and Snail were the functional targets of miR-22. Furthermore, MALAT1 could modulate MMP14 and Snail by operating as a competing endogenous RNA (ceRNA) for miR-22. The effects of MALAT1 in malignant melanoma is verified using a xenograft model. This finding elucidates a new mechanism for MALAT1 in melanoma development and provides a potential target for melanoma therapeutic intervention.

Effects of dehydroepiandrosterone in rats injected with streptozotocin during the neonatal period.

PubMed

Giroix, M H; Malaisse-Lagae, F; Portha, B; Sener, A; Malaisse, W J

1997-06-01

Control rats and diabetic animals injected with streptozotocin during the neonatal period were either maintained on a standard diet or given access to food supplemented with dehydroepiandrosterone (DHEA, 0.2%) for 11 days before sacrifice. In both control and diabetic rats, DHEA feeding augmented the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase and cytosolic NADP-linked malate dehydrogenase in liver, but not so in either the parotid gland or pancreatic islets. DHEA lowered, in both control and diabetic rats, the ratio between D-glucose oxidation and utilization and the rate of insulin release in pancreatic islets exposed to a high concentration of D-glucose, as well as the insulin concentration and insulin/glucose ratio in plasma. These findings support the view that, in diabetes, DHEA, by increasing sensitivity to insulin, may allow islet B-cells to avoid the otherwise unfavorable consequences of chronic hyperactivity.

Metagenomic analysis of rumen microbial population in dairy heifers fed a high grain diet supplemented with dicarboxylic acids or polyphenols.

PubMed

De Nardi, Roberta; Marchesini, Giorgio; Li, Shucong; Khafipour, Ehsan; Plaizier, Kees J C; Gianesella, Matteo; Ricci, Rebecca; Andrighetto, Igino; Segato, Severino

2016-02-19

The aim of this study was to investigate the effects of two feed supplements on rumen bacterial communities of heifers fed a high grain diet. Six Holstein-Friesian heifers received one of the following dietary treatments according to a Latin square design: no supplement (control, C), 60 g/day of fumarate-malate (organic acid, O) and 100 g/day of polyphenol-essential oil (P). Rumen fluid was analyzed to assess the microbial population using Illumina sequencing and quantitative real time PCR. The P treatment had the highest number of observed species (P < 0.10), Chao1 index (P < 0.05), abundance based coverage estimated (ACE) (P < 0.05), and Fisher's alpha diversity (P < 0.10). The O treatment had intermediate values between C and P treatments with the exception of the Chao1 index. The PCoA with unweighted Unifrac distance showed a separation among dietary treatments (P = 0.09), above all between the C and P (P = 0.05). The O and P treatments showed a significant increase of the family Christenenellaceae and a decline of Prevotella brevis compared to C. Additionally, the P treatment enhanced the abundance of many taxa belonging to Bacteroidetes, Firmicutes and Tenericutes phyla due to a potential antimicrobial activity of flavonoids that increased competition among bacteria. Organic acid and polyphenols significantly modified rumen bacterial populations during high-grain feeding in dairy heifers. In particular the polyphenol treatment increased the richness and diversity of rumen microbiota, which are usually high in conditions of physiological rumen pH and rumen function.

Malate secretion from the root system is an important reason for higher resistance of Miscanthus sacchariflorus to cadmium.

PubMed

Guo, Haipeng; Feng, Xue; Hong, Chuntao; Chen, Houming; Zeng, Fanrong; Zheng, Bingsong; Jiang, Dean

2017-03-01

Miscanthus is a vigorous perennial Gramineae genus grown throughout the world as a promising bioenergy crop and generally regarded as heavy metal tolerant due to its ability to absorb heavy metals. However, little is known about the mechanism for heavy metal tolerance in Miscanthus. In this study, two Miscanthus species (Miscanthus sacchariflorus and Miscanthus floridulus) exhibiting different cadmium (Cd) sensitivity were used to address the mechanisms of Cd tolerance. Under the same Cd stress, M. sacchariflorus showed higher Cd tolerance with better growth and lower Cd accumulation in both shoots and roots than M. floridulus. The malate (MA) content significantly increased in root exudates of M. sacchariflorus following Cd treatment while it was almost unchanged in M. floridulus. Cellular Cd analysis and flux data showed that exogenous MA application markedly restricted Cd influx and accumulation while an anion-channel inhibitor (phenylglyoxal) effectively blocked Cd-induced MA secretion and increased Cd influx in M. sacchariflorus, indicating that MA secretion could alleviate Cd toxicity by reducing Cd uptake. The genes of malate dehydrogenases (MsMDHs) and Al-activated malate transporter 1 (MsALMT1) in M. sacchariflorus were highly upregulated under Cd stress, compared with that in M. floridulus. The results indicate that Cd-induced MA synthesis and secretion efficiently alleviate Cd toxicity by reducing Cd influx in M. sacchariflorus. © 2016 Scandinavian Plant Physiology Society.

Novel properties of the wheat aluminum tolerance organic acid transporter (TaALMT1) revealed by electrophysiological characterization in Xenopus Oocytes: functional and structural implications.

PubMed

Piñeros, Miguel A; Cançado, Geraldo M A; Kochian, Leon V

2008-08-01

Many plant species avoid the phytotoxic effects of aluminum (Al) by exuding dicarboxylic and tricarboxylic acids that chelate and immobilize Al(3+) at the root surface, thus preventing it from entering root cells. Several novel genes that encode membrane transporters from the ALMT and MATE families recently were cloned and implicated in mediating the organic acid transport underlying this Al tolerance response. Given our limited understanding of the functional properties of ALMTs, in this study a detailed characterization of the transport properties of TaALMT1 (formerly named ALMT1) from wheat (Triticum aestivum) expressed in Xenopus laevis oocytes was conducted. The electrophysiological findings are as follows. Although the activity of TaALMT1 is highly dependent on the presence of extracellular Al(3+) (K(m1/2) of approximately 5 microm Al(3+) activity), TaALMT1 is functionally active and can mediate ion transport in the absence of extracellular Al(3+). The lack of change in the reversal potential (E(rev)) upon exposure to Al(3+) suggests that the "enhancement" of TaALMT1 malate transport by Al is not due to alteration in the transporter's selectivity properties but is solely due to increases in its anion permeability. The consistent shift in the direction of the E(rev) as the intracellular malate activity increases indicates that TaALMT1 is selective for the transport of malate over other anions. The estimated permeability ratio between malate and chloride varied between 1 and 30. However, the complex behavior of the E(rev) as the extracellular Cl(-) activity was varied indicates that this estimate can only be used as a general guide to understanding the relative affinity of TaALMT1 for malate, representing only an approximation of those expected under physiologically relevant ionic conditions. TaALMT1 can also mediate a large anion influx (i.e. outward currents). TaALMT1 is permeable not only to malate but also to other physiologically relevant anions such as Cl(-), NO(3)(-), and SO(4)(2-) (to a lesser degree).

Novel Properties of the Wheat Aluminum Tolerance Organic Acid Transporter (TaALMT1) Revealed by Electrophysiological Characterization in Xenopus Oocytes: Functional and Structural Implications1[OA

PubMed Central

Piñeros, Miguel A.; Cançado, Geraldo M.A.; Kochian, Leon V.

2008-01-01

Many plant species avoid the phytotoxic effects of aluminum (Al) by exuding dicarboxylic and tricarboxylic acids that chelate and immobilize Al3+ at the root surface, thus preventing it from entering root cells. Several novel genes that encode membrane transporters from the ALMT and MATE families recently were cloned and implicated in mediating the organic acid transport underlying this Al tolerance response. Given our limited understanding of the functional properties of ALMTs, in this study a detailed characterization of the transport properties of TaALMT1 (formerly named ALMT1) from wheat (Triticum aestivum) expressed in Xenopus laevis oocytes was conducted. The electrophysiological findings are as follows. Although the activity of TaALMT1 is highly dependent on the presence of extracellular Al3+ (Km1/2 of approximately 5 μm Al3+ activity), TaALMT1 is functionally active and can mediate ion transport in the absence of extracellular Al3+. The lack of change in the reversal potential (Erev) upon exposure to Al3+ suggests that the “enhancement” of TaALMT1 malate transport by Al is not due to alteration in the transporter's selectivity properties but is solely due to increases in its anion permeability. The consistent shift in the direction of the Erev as the intracellular malate activity increases indicates that TaALMT1 is selective for the transport of malate over other anions. The estimated permeability ratio between malate and chloride varied between 1 and 30. However, the complex behavior of the Erev as the extracellular Cl− activity was varied indicates that this estimate can only be used as a general guide to understanding the relative affinity of TaALMT1 for malate, representing only an approximation of those expected under physiologically relevant ionic conditions. TaALMT1 can also mediate a large anion influx (i.e. outward currents). TaALMT1 is permeable not only to malate but also to other physiologically relevant anions such as Cl−, NO3−, and SO42− (to a lesser degree). PMID:18550686

Long non-coding RNA MALAT1 for promoting metastasis and proliferation by acting as a ceRNA of miR-144-3p in osteosarcoma cells

PubMed Central

Wang, Ningning; Li, Pengcheng; Zeng, Xiandong; Zhang, Weiguo

2017-01-01

Long non-coding RNAs (lncRNAs) are involved in various biological processes and diseases including osteosarcoma. Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is overly expressed in osteosarcoma. But the function and mechanism it works on in osteosarcoma proliferation and metastasis mediated by Rho associated coiled-coil containing protein kinase 1 (ROCK1) and Rho associated coiled-coil containing protein kinase 2 (ROCK2) remain unclear. In the present study, an elevated MALAT1 was found in osteosarcoma tissues and cell lines, and the elevated MALAT1 was correlated with a poor prognosis in osteosarcoma patients. The functional experiments show that a decreased MALAT1 could remarkably inhibit osteosarcoma cell metastasis and proliferation but induce cell cycle arrest, indicating that MALAT1 functioned as an oncogene in osteosarcoma. Furthermore, we confirmed that MALAT1 and ROCK1/ROCK2 which were targeted by microRNA-144-3p (miR-144-3p) shared the same miR-144-3p combining site. Furthermore, the constructed luciferase assay verified that MALAT1 was a target of miR-144-3p. Additionally, the results of a qRT-PCR demonstrated that MALAT1 and miR-144-3p repressed each other's expression in a reciprocal manner. Finally, we affirmed that an overexpression of MALAT1 inhibited ROCK1/ROCK2 expression and its mediated metastasis and proliferation by working as a competitive endogenous RNA (ceRNA) via miR-144-3p. In summary, the findings of this study based on the ceRNA theory, combining the research foundation of miR-144-3p, ROCK1 and ROCK2, taking MALAT1 as a new point of study, provided new insights into molecular level proliferation reversal and metastasis of osteosarcoma. PMID:28938647

Citrate, malate and alkali content in commonly consumed diet sodas: implications for nephrolithiasis treatment.

PubMed

Eisner, Brian H; Asplin, John R; Goldfarb, David S; Ahmad, Ardalanejaz; Stoller, Marshall L

2010-06-01

Citrate is a known inhibitor of calcium stone formation. Dietary citrate and alkali intake may have an effect on citraturia. Increasing alkali intake also increases urine pH, which can help prevent uric acid stones. We determined citrate, malate and total alkali concentrations in commonly consumed diet sodas to help direct dietary recommendations in patients with hypocitraturic calcium or uric acid nephrolithiasis. Citrate and malate were measured in a lemonade beverage commonly used to treat hypocitraturic calcium nephrolithiasis and in 15 diet sodas. Anions were measured by ion chromatography. The pH of each beverage was measured to allow calculation of the unprotonated anion concentration using the known pK of citric and malic acid. Total alkali equivalents were calculated for each beverage. Statistical analysis was done using Pearson's correlation coefficient. Several sodas contained an amount of citrate equal to or greater than that of alkali and total alkali as a lemonade beverage commonly used to treat hypocitraturic calcium nephrolithiasis (6.30 mEq/l citrate as alkali and 6.30 as total alkali). These sodas were Diet Sunkist Orange, Diet 7Up, Sprite Zero, Diet Canada Dry Ginger Ale, Sierra Mist Free, Diet Orange Crush, Fresca and Diet Mountain Dew. Colas, including Caffeine Free Diet Coke, Coke Zero, Caffeine Free Diet Pepsi and Diet Coke with Lime, had the lowest total alkali (less than 1.0 mEq/l). There was no significant correlation between beverage pH and total alkali content. Several commonly consumed diet sodas contain moderate amounts of citrate as alkali and total alkali. This information is helpful for dietary recommendations in patients with calcium nephrolithiasis, specifically those with hypocitraturia. It may also be useful in patients with low urine pH and uric acid stones. Beverage malate content is also important since malate ingestion increases the total alkali delivered, which in turn augments citraturia and increases urine pH. Copyright 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Functional and prognostic significance of long non-coding RNA MALAT1 as a metastasis driver in ER negative lymph node negative breast cancer

PubMed Central

Jadaliha, Mahdieh; Zong, Xinying; Malakar, Pushkar; Ray, Tania; Singh, Deepak K.; Freier, Susan M.; Jensen, Tor; Prasanth, Supriya G.; Karni, Rotem; Ray, Partha S.; Prasanth, Kannanganattu V.

2016-01-01

MALAT1 (metastasis associated lung adenocarcinoma transcript1) is a conserved long non-coding RNA, known to regulate gene expression by modulating transcription and post-transcriptional pre-mRNA processing of a large number of genes. MALAT1 expression is deregulated in various tumors, including breast cancer. However, the significance of such abnormal expression is yet to be fully understood. In this study, we demonstrate that regulation of aggressive breast cancer cell traits by MALAT1 is not predicted solely based on an elevated expression level but is context specific. By performing loss- and gain-of-function studies, both under in vitro and in vivo conditions, we demonstrate that MALAT1 facilitates cell proliferation, tumor progression and metastasis of triple-negative breast cancer (TNBC) cells despite having a comparatively lower expression level than ER or HER2-positive breast cancer cells. Furthermore, MALAT1 regulates the expression of several cancer metastasis-related genes, but displays molecular subtype specific correlations with such genes. Assessment of the prognostic significance of MALAT1 in human breast cancer (n=1992) revealed elevated MALAT1 expression was associated with decreased disease-specific survival in ER negative, lymph node negative patients of the HER2 and TNBC molecular subtypes. Multivariable analysis confirmed MALAT1 to have independent prognostic significance in the TNBC lymph node negative patient subset (HR=2.64, 95%CI 1.35 − 5.16, p=0.005). We propose that the functional significance of MALAT1 as a metastasis driver and its potential use as a prognostic marker is most promising for those patients diagnosed with ER negative, lymph node negative breast cancer who might otherwise mistakenly be stratified to have low recurrence risk. PMID:27250026

Functional and prognostic significance of long non-coding RNA MALAT1 as a metastasis driver in ER negative lymph node negative breast cancer.

PubMed

Jadaliha, Mahdieh; Zong, Xinying; Malakar, Pushkar; Ray, Tania; Singh, Deepak K; Freier, Susan M; Jensen, Tor; Prasanth, Supriya G; Karni, Rotem; Ray, Partha S; Prasanth, Kannanganattu V

2016-06-28

MALAT1 (metastasis associated lung adenocarcinoma transcript1) is a conserved long non-coding RNA, known to regulate gene expression by modulating transcription and post-transcriptional pre-mRNA processing of a large number of genes. MALAT1 expression is deregulated in various tumors, including breast cancer. However, the significance of such abnormal expression is yet to be fully understood. In this study, we demonstrate that regulation of aggressive breast cancer cell traits by MALAT1 is not predicted solely based on an elevated expression level but is context specific. By performing loss- and gain-of-function studies, both under in vitro and in vivo conditions, we demonstrate that MALAT1 facilitates cell proliferation, tumor progression and metastasis of triple-negative breast cancer (TNBC) cells despite having a comparatively lower expression level than ER or HER2-positive breast cancer cells. Furthermore, MALAT1 regulates the expression of several cancer metastasis-related genes, but displays molecular subtype specific correlations with such genes. Assessment of the prognostic significance of MALAT1 in human breast cancer (n=1992) revealed elevated MALAT1 expression was associated with decreased disease-specific survival in ER negative, lymph node negative patients of the HER2 and TNBC molecular subtypes. Multivariable analysis confirmed MALAT1 to have independent prognostic significance in the TNBC lymph node negative patient subset (HR=2.64, 95%CI 1.35- 5.16, p=0.005). We propose that the functional significance of MALAT1 as a metastasis driver and its potential use as a prognostic marker is most promising for those patients diagnosed with ER negative, lymph node negative breast cancer who might otherwise mistakenly be stratified to have low recurrence risk.

Tricarboxylic acid cycle without malate dehydrogenase in Streptomyces coelicolor M-145.

PubMed

Takahashi-Íñiguez, Tóshiko; Barrios-Hernández, Joana; Rodríguez-Maldonado, Marion; Flores, María Elena

2018-06-23

The oxidation of malate to oxaloacetate is catalysed only by a nicotinamide adenine dinucleotide-dependent malate dehydrogenase encoded by SCO4827 in Streptomyces coelicolor. A mutant lacking the malate dehydrogenase gene was isolated and no enzymatic activity was detected. As expected, the ∆mdh mutant was unable to grow on malate as the sole carbon source. However, the mutant grew less in minimal medium with glucose and there was a delay of 36 h. The same behaviour was observed when the mutant was grown on minimal medium with casamino acids or glycerol. For unknown reasons, the mutant was not able to grow in YEME medium with glucose. The deficiency of malate dehydrogenase affected the expression of the isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase genes, decreasing the expression of both genes by approximately two- to threefold.

Serum long non coding RNA MALAT-1 protected by exosomes is up-regulated and promotes cell proliferation and migration in non-small cell lung cancer.

PubMed

Zhang, Rui; Xia, Yuhong; Wang, Zhixin; Zheng, Jie; Chen, Yafei; Li, Xiaoli; Wang, Yu; Ming, Huaikun

2017-08-19

Circulating lncRNAs have been defined as a novel biomarker for non-small cell lung cancer (NSCLC), MALAT-1 was first identified lncRNA that was related to lung cancer metastasis. However, the relationship between exosomal lncRNAs and the diagnosis and prognosis of NSCLC was poorly understood. The aim of this study is to evaluate the clinical significance of serum exosomal MALAT-1 as a biomarker in the metastasis of NSCLC. In this study, we firstly isolated the exosomes from healthy subjects and NSCLC patients. Then we measured the expression levels of MALAT-1 contained in exosomes, and found that exosomal MALAT-1 was highly expressed in NSCLC patients, more importantly, the levels of exosomal MALAT-1 were positively associated with tumor stage and lymphatic metastasis. In addition, we decreased MALAT-1 expression by short hairpin RNA and conducted a series of assays including MTT, cell cycle, colony formation, wound-healing scratch and Annexin/V PI by flow cytometry in human lung cancer cell lines. These in vitro studies demonstrated that serum exosome-derived long noncoding RNA MALAT-1 promoted the tumor growth and migration, and prevented tumor cells from apoptosis in lung cancer cell lines. Taken together, this study shed a light on utilizing MALAT-1 in exosomes as a non-invasive serum-based tumor biomarker for diagnosis and prognosis of NSCLC. Copyright © 2017 Elsevier Inc. All rights reserved.

Leaf malate and succinate accumulation are out of phase throughout the development of the CAM plant Ananas comosus.

PubMed

Rainha, N; Medeiros, V P; Ferreira, C; Raposo, A; Leite, J P; Cruz, C; Pacheco, C A; Ponte, D; Silva, A B

2016-03-01

In plants with Crassulacean Acid Metabolism (CAM), organic acids, mainly malate are crucial intermediates for carbon fixation. In this research we studied the circadian oscillations of three organic anions (malate, citrate, and succinate) in Ananas comosus, assessing the effect of season and plant development stage. Seasonal and plant development dependencies were observed. The circadian oscillations of malate and citrate were typical of CAM pathways reported in the literature. Citrate content was quite stable (25-30 μmol g(-1) FW) along the day, with a seasonal effect. Succinate was shown to have both diurnal and seasonal oscillations and also a correlation with malate, since it accumulated during the afternoon when malate content was normally at a minimum, suggesting a possible mechanistic effect between both anions in CAM and/or respiratory metabolisms. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Cellular distribution, purification and electrophoretic properties of malate dehydrogenase in Trichuris ovis and inhibition by benzimidazoles and pyrimidine derivatives.

PubMed

Sanchez-Moreno, M; Ortega, J E; Valero, A

1989-12-01

High levels of malate dehydrogenase were found in Trichuris ovis. Two molecular forms of the enzyme, of different cellular location and electrophoretic pattern, were isolated and purified. The activity of soluble malate dehydrogenase was greater than that of mitochondrial malate dehydrogenase. Both forms also displayed different electrophoretic profiles in comparison with purified extracts from goat (Capra hircus) liver. Substrate concentration directly affected enzyme activity. Host and parasite malate dehydrogenase activity were both inhibited by a series of benzimidazoles and pyrimidine-derived compounds, some of which markedly reduced parasite enzyme activity, but not host enzyme activity. Percentage inhibition by some pyrimidine derivatives was greater than that produced by benzimidazoles.

Development and prospective multicenter evaluation of the long noncoding RNA MALAT-1 as a diagnostic urinary biomarker for prostate cancer

PubMed Central

Lu, Ji; Shi, Xiaolei; Zhu, Yasheng; Zhang, Wei; Jing, Taile; Zhang, Chao; Shen, Jian; Xu, Chuanliang; Wang, Huiqing; Wang, Haifeng; Wang, Yang; Liu, Bin; Li, Yaoming; Fang, Ziyu; Guo, Fei; Qiao, Meng; Wu, Chengyao; Wei, Qiang; Xu, Danfeng; Shen, Dan; Lu, Xin; Gao, Xu; Hou, Jianguo; Sun, Yinghao

2014-01-01

The current strategy for diagnosing prostate cancer (PCa) is mainly based on the serum prostate-specific antigen (PSA) test. However, PSA has low specificity and has led to numerous unnecessary biopsies. We evaluated the effectiveness of urinary metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), a long noncoding RNA, for predicting the risk of PCa before biopsy. The MALAT-1 score was tested in a discovery phase and a multi-center validation phase. The predictive power of the MALAT-1 score was evaluated by the area under receiver operating characteristic (ROC) curve (AUC) and by decision curve analysis. As an independent predictor of PCa, the MALAT-1 score was significantly higher in men with a positive biopsy than in those with a negative biopsy. The ROC analysis showed a higher AUC for the MALAT-1 score (0.670 and 0.742) vs. the total PSA (0.545 and 0.601) and percent free PSA (0.622 and 0.627) in patients with PSA values of 4.0-10 ng/ml. According to the decision curve analysis, using a probability threshold of 25%, the MALAT-1 model would prevent 30.2%-46.5% of unnecessary biopsies in PSA 4–10 ng/ml cohorts, without missing any high-grade cancers. Our results demonstrate that urine MALAT-1 is a promising biomarker for predicting prostate cancer risk. PMID:25526029

Identification and characterization of proliferative retinopathy-related long noncoding RNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Rong-Mei; Wang, Xiao-Qun; Yao, Jin

2015-09-25

Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment and vitreoretinal surgery, which can lead to severe vision reduction. Long non-coding RNAs (lncRNAs) play critical roles in many biological processes and disease development. We attempted to determine the role of lncRNAs in the setting of PVR. Microarray analysis revealed that 78 lncRNAs were abnormally expressed in the epiretinal membranes (ERMs) of PVR patients, including 48 up-regulated and 30 down-regulated lncRNA transcripts. We subsequently focus on one lncRNA, MALAT1, and investigated its expression pattern in the biofluid of PVR patients. MALAT1 was significantly up-regulated in the cellular and plasma fractionmore » of peripheral blood in PVR patients. MALAT1 expression was obviously reduced after PVR operation. In vitro experiments revealed the role of MALAT1 in regulating RPE proliferation and migration, which is critical for ERMs formation. This study suggests that lncRNAs are the potential regulators of PVR pathology. MALAT1 is a potential prognostic indicator and a target for the diagnosis and gene therapy for PVR diseases. - Highlights: • 78 lncRNAs are differentially expressed between PVR-ERMs and secondary ERMs. • MALAT1 level is elevated in the ERMs of PVR patients. • Circulating MALAT1 level is up-regulated in PVR patients. • MALAT1 knockdown regulates RPE proliferation and migration.« less

Long non-coding RNA MALAT1 modulates radiosensitivity of HR-HPV+ cervical cancer via sponging miR-145.

PubMed

Lu, Hongzhi; He, Yu; Lin, Lin; Qi, Zhengqin; Ma, Li; Li, Li; Su, Ying

2016-02-01

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a lncRNA playing oncogenic role in several cancers, including cervical cancer. However, its role in radiosensitivity of cervical cancer is not yet well understood. This study explored the role of MALAT1 in radiosensitivity of high-risk human papillomavirus (HR-HPV)-positive cervical cancer and whether there is a ceRNA mechanism which participated in its regulation over radiosensitivity. Based on tissue samples from 50 cervical cancer cases and 25 healthy controls, we found MALAT1 expression was significantly higher in radioresistant than in radiosensitive cancer cases. In addition, MALAT1 and miR-145 expression inversely changed in response to irradiation in HR-HPV+ cervical cancer cells. By using clonogenic assay and flow cytometry analysis of cell cycle distribution and apoptosis, we found CaSki and Hela cells with knockdown of MALAT1 had significantly lower colony formation, higher ratio of G2/M phase block and higher ratio of cell apoptosis. By performing RNA-binding protein immunoprecipitation (RIP) assay and RNA pull-down assay, we confirmed that miR-145 and MALAT1 were in the same Ago2 complex and there was a reciprocal repression between them. Then, we explored the function of MALAT1-miR-145 in radiosensitivity of cervical cancers cells and demonstrated that si-MALAT1 and miR-145 had some level of synergic effect in reducing cancer cell colony formation, cell cycle regulation, and inducing apoptosis. These findings provide an important clue about microRNA-lncRNA interaction in the mechanism of radioresistance of cervical cancer.

Factors affecting the translocation of oxaloacetate and l-malate into rat liver mitochondria

PubMed Central

Haslam, J. M.; Griffiths, D. E.

1968-01-01

1. The rates of translocation of oxaloacetate and l-malate into rat liver mitochondria were measured by a direct spectrophotometric assay. 2. Penetration obeyed Michaelis–Menten kinetics, and apparent Km values were 40μm for oxaloacetate and 0·13mm for l-malate. 3. Arrhenius plots of the temperature-dependence of rates of penetration gave activation energies of +10kcal./mole for oxaloacetate and +8kcal./mole for l-malate. 4. The translocation of both oxaloacetate and l-malate was competitively inhibited by d-malate, succinate, malonate, meso-tartrate, maleate and citraconate. The Ki values of these inhibitors were similar for the penetration of both oxaloacetate and l-malate. 5. Rates of penetration were stimulated by NNN′N′-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate under aerobic conditions or by ATP under anaerobic conditions. 6. The energy-dependent stimulation of translocation was abolished by uncouplers of oxidative phosphorylation. Oligomycin A, aurovertin, octyl-guanidine and atractyloside prevented the stimulation by ATP, but did not inhibit the stimulation by NNN′N′-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate. 7. Mitochondria prepared in the presence of ethylene-dioxybis(ethyleneamino)tetra-acetic acid did not exhibit the energy-dependent translocation, but this could be restored by the addition of 50μm-calcium chloride. 8. Valinomycin or gramicidin plus potassium chloride enhanced the energy-dependent translocation of oxaloacetate and l-malate. 9. Addition of oxaloacetate stimulated the adenosine triphosphatase activity of the mitochondria, and the ratio of `extra' oxaloacetate translocation to `extra' adenosine triphosphatase activity was 1·6:1. 10. Possible mechanisms for the energy-dependent entry of oxaloacetate and l-malate into mitochondria are discussed in relation to the above results. PMID:4235143

Medium-chain triglycerides supplement therapy with a low-carbohydrate formula can supply energy and enhance ammonia detoxification in the hepatocytes of patients with adult-onset type II citrullinemia.

PubMed

Hayasaka, Kiyoshi; Numakura, Chikahiko; Yamakawa, Mitsunori; Mitsui, Tetsuo; Watanabe, Hisayoshi; Haga, Hiroaki; Yazaki, Masahide; Ohira, Hiromasa; Ochiai, Yasuo; Tahara, Toshiyuki; Nakahara, Tamio; Yamashiki, Noriyo; Nakayama, Takahiro; Kon, Takashi; Mitsubuchi, Hiroshi; Yoshida, Hiroshi

2018-04-12

Citrin, encoded by SLC25A13, constitutes the malate-aspartate shuttle, the main NADH-shuttle in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). Citrin deficiency is predicted to impair hepatic glycolysis and de novo lipogenesis, resulting in hepatic energy deficit. Secondary decrease in hepatic argininosuccinate synthetase (ASS1) expression has been considered a cause of hyperammonemia in CTLN2. We previously reported that medium-chain triglyceride (MCT) supplement therapy with a low-carbohydrate formula was effective in CTLN2 to prevent a relapse of hyperammonemic encephalopathy. We present the therapy for six CTLN2 patients. All the patients' general condition steadily improved and five patients with hyperammonemic encephalopathy recovered from unconsciousness in a few days. Before the treatment, plasma glutamine levels did not increase over the normal range and rather decreased to lower than the normal range in some patients. The treatment promptly decreased the blood ammonia level, which was accompanied by a decrease in plasma citrulline levels and an increase in plasma glutamine levels. These findings indicated that hyperammonemia was not only caused by the impairment of ureagenesis at ASS1 step, but was also associated with an impairment of glutamine synthetase (GS) ammonia-detoxification system in the hepatocytes. There was no decrease in the GS expressing hepatocytes. MCT supplement with a low-carbohydrate formula can supply the energy and/or substrates for ASS1 and GS, and enhance ammonia detoxification in hepatocytes. Histological improvement in the hepatic steatosis and ASS1-expression was also observed in a patient after long-term treatment.

The contribution of stored malate and citrate to the substrate requirements of metabolism of ripening peach (Prunus persica L. Batsch) flesh is negligible. Implications for the occurrence of phosphoenolpyruvate carboxykinase and gluconeogenesis.

PubMed

Famiani, Franco; Farinelli, Daniela; Moscatello, Stefano; Battistelli, Alberto; Leegood, Richard C; Walker, Robert P

2016-04-01

The first aim of this study was to determine the contribution of stored malate and citrate to the substrate requirements of metabolism in the ripening flesh of the peach (Prunus persica L. Batsch) cultivar Adriatica. In the flesh, stored malate accumulated before ripening could contribute little or nothing to the net substrate requirements of metabolism. This was because there was synthesis and not dissimilation of malate throughout ripening. Stored citrate could potentially contribute a very small amount (about 5.8%) of the substrate required by metabolism when the whole ripening period was considered, and a maximum of about 7.5% over the latter part of ripening. The second aim of this study was to investigate why phosphoenolpyruvate carboxykinase (PEPCK) an enzyme utilised in gluconeogenesis from malate and citrate is present in peach flesh. The occurrence and localisation of enzymes utilised in the metabolism of malate, citrate and amino acids were determined in peach flesh throughout its development. Phosphoenolpyruvate carboxylase (essential for the synthesis of malate and citrate) was present in the same cells and at the same time as PEPCK and NADP-malic enzyme (both utilised in the dissimilation of malate and citrate). A hypothesis is presented to explain the presence of these enzymes and to account for the likely occurrence of gluconeogenesis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Long non-coding RNA MALAT1 interacts with transcription factor Foxo1 to regulate SIRT1 transcription in high glucose-induced HK-2 cells injury.

PubMed

Zhou, Ling; Xu, De-Yu; Sha, Wen-Gang; Shen, Lei; Lu, Guo-Yuan

2018-06-18

Tubular injury is considered as a crucial pathological feature of diabetic nephropathy. LncRNA MALAT1 is involved in diabetic complications. Hence the role of MALAT1 in high glucose-induced renal tubular epithelial cells (HK-2) injury deserves investigation. The diabetic mice model was established with streptozotocin (STZ) injection. The expression of NEAT1, SIRT1, and Foxo1 mRNA and protein was determined with qRT-PCR and western blot, respectively. The serum creatinine and urinary albumin were examined by enzyme linked immunosorbent assay (ELISA). Interaction between MALAT1 and Foxo1 was detected with RIP and RNA pull-down assay, respectively. Dual luciferase reporter assay was used to evaluate the binding between Foxo1 and SIRT1. LncRNA MALAT1 was up-regulated in kidney tissues of diabetic mice and in HK-2 cells treated with high glucose, while the expression of SIRT1 was decreased. Interaction between MALAT1 and Foxo1 was observed in HK-2 cells and the interaction was promoted by high glucose treatment. Foxo1 activated SIRT1 transcription by binding to its promoter, and MALAT1 repressed SIRT1 expression through targeting Foxo1. LncRNA MALAT1 interacts with transcription factor Foxo1 to represses SIRT1 transcription in high glucose incubated HK-2 cells, which promotes high glucose-induced HK-2 cells injury. Copyright © 2018. Published by Elsevier Inc.

Creatine and creatine forms intended for sports nutrition.

PubMed

Andres, Susanne; Ziegenhagen, Rainer; Trefflich, Iris; Pevny, Sophie; Schultrich, Katharina; Braun, Hans; Schänzer, Wilhelm; Hirsch-Ernst, Karen Ildico; Schäfer, Bernd; Lampen, Alfonso

2017-06-01

Creatine is a popular ergogenic supplement in sports nutrition. Yet, supplementation of creatine occasionally caused adverse effects such as gastrointestinal complaints, muscle cramps and an increase in body weight. Creatine monohydrate has already been evaluated by different competent authorities and several have come to the conclusion that a daily intake of 3 g creatine per person is unlikely to pose safety concerns, focusing on healthy adults with exclusion of pregnant and breastfeeding women. Possible vulnerable subgroups were also discussed in relation to the safety of creatine. The present review provides an up-to-date overview of the relevant information with special focus on human studies regarding the safety of creatine monohydrate and other marketed creatine forms, in particular creatine pyruvate, creatine citrate, creatine malate, creatine taurinate, creatine phosphate, creatine orotate, creatine ethyl ester, creatine pyroglutamate, creatine gluconate, and magnesium creatine chelate. Limited data are available with regard to the safety of the latter creatine forms. Considering an acceptable creatine intake of 3 g per day, most of the evaluated creatine forms are unlikely to pose safety concerns, however some safety concerns regarding a supplementary intake of creatine orotate, creatine phosphate, and magnesium creatine chelate are discussed here. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

C-terminus-mediated voltage gating of Arabidopsis guard cell anion channel QUAC1.

PubMed

Mumm, Patrick; Imes, Dennis; Martinoia, Enrico; Al-Rasheid, Khaled A S; Geiger, Dietmar; Marten, Irene; Hedrich, Rainer

2013-09-01

Anion transporters in plants play a fundamental role in volume regulation and signaling. Currently, two plasma membrane-located anion channel families—SLAC/SLAH and ALMT—are known. Among the ALMT family, the root-expressed ALuminium-activated Malate Transporter 1 was identified by comparison of aluminum-tolerant and Al(3+)-sensitive wheat cultivars and was subsequently shown to mediate voltage-independent malate currents. In contrast, ALMT12/QUAC1 (QUickly activating Anion Channel1) is expressed in guard cells transporting malate in an Al(3+)-insensitive and highly voltage-dependent manner. So far, no information is available about the structure and mechanism of voltage-dependent gating with the QUAC1 channel protein. Here, we analyzed gating of QUAC1-type currents in the plasma membrane of guard cells and QUAC1-expressing oocytes revealing similar voltage dependencies and activation–deactivation kinetics. In the heterologous expression system, QUAC1 was electrophysiologically characterized at increasing extra- and intracellular malate concentrations. Thereby, malate additively stimulated the voltage-dependent QUAC1 activity. In search of structural determinants of the gating process, we could not identify transmembrane domains common for voltage-sensitive channels. However, site-directed mutations and deletions at the C-terminus of QUAC1 resulted in altered voltage-dependent channel activity. Interestingly, the replacement of a single glutamate residue, which is conserved in ALMT channels from different clades, by an alanine disrupted QUAC1 activity. Together with C- and N-terminal tagging, these results indicate that the cytosolic C-terminus is involved in the voltage-dependent gating mechanism of QUAC1.

Long noncoding RNA MALAT1 enhances the docetaxel resistance of prostate cancer cells via miR-145-5p-mediated regulation of AKAP12.

PubMed

Xue, Dong; Lu, Hao; Xu, Han-Yan; Zhou, Cui-Xing; He, Xiao-Zhou

2018-06-01

Our present work was aimed to study on the regulatory role of MALAT1/miR-145-5p/AKAP12 axis on docetaxel (DTX) sensitivity of prostate cancer (PCa) cells. The microarray data (GSE33455) to identify differentially expressed lncRNAs and mRNAs in DTX-resistant PCa cell lines (DU-145-DTX and PC-3-DTX) was retrieved from the Gene Expression Omnibus (GEO) database. QRT-PCR analysis was performed to measure MALAT1 expression in DTX-sensitive and DTX-resistant tissues/cells. The human DTX-resistant cell lines DU145-PTX and PC3-DTX were established as in vitro cell models, and the expression of MALAT1, miR-145-5p and AKAP12 was manipulated in DTX-sensitive and DTX-resistant cells. Cell viability was examined using MTT assay and colony formation methods. Cell apoptosis was assessed by TUNEL staining. Cell migration and invasion was determined by scratch test (wound healing) and Transwell assay, respectively. Dual-luciferase assay was applied to analyse the target relationship between lncRNA MALAT1 and miR-145-5p, as well as between miR-145-5p and AKAP12. Tumour xenograft study was undertaken to confirm the correlation of MALAT1/miR-145-5p/AKAP12 axis and DTX sensitivity of PCa cells in vivo. In this study, we firstly notified that the MALAT1 expression levels were up-regulated in clinical DTX-resistant PCa samples. Overexpressed MALAT1 promoted cell proliferation, migration and invasion but decreased cell apoptosis rate of PCa cells in spite of DTX treatment. We identified miR-145-5p as a target of MALAT1. MiR-145-5p overexpression in PC3-DTX led to inhibited cell proliferation, migration and invasion as well as reduced chemoresistance to DTX, which was attenuated by MALAT1. Moreover, we determined that AKAP12 was a target of miR-145-5p, which significantly induced chemoresistance of PCa cells to DTX. Besides, it was proved that MALAT1 promoted tumour cell proliferation and enhanced DTX-chemoresistance in vivo. There was an lncRNA MALAT1/miR-145-5p/AKAP12 axis involved in DTX resistance of PCa cells and provided a new thought for PCa therapy. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Materials and methods for efficient succinate and malate production

DOEpatents

Jantama, Kaemwich; Haupt, Mark John; Zhang, Xueli; Moore, Jonathan C; Shanmugam, Keelnatham T; Ingram, Lonnie O'Neal

2014-04-08

Genetically engineered microorganisms have been constructed to produce succinate and malate in mineral salt media in pH-controlled batch fermentations without the addition of plasmids or foreign genes. The subject invention also provides methods of producing succinate and malate comprising the culture of genetically modified microorganisms.

Characterization of the N-Acetyl-[alpha]-d-glucosaminyl l-Malate Synthase and Deacetylase Functions for Bacillithiol Biosynthesis in Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parsonage, Derek; Newton, Gerald L.; Holder, Robert C.

2012-02-21

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce {alpha}-D-glucosaminyl L-malate (GlcN-malate) from UDP-GlcNAc and L-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase ({yields}GlcNAc-malate) and the BaBshB deacetylase ({yields}GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternarymore » complex, determined in this work at 3.3 {angstrom} resolution, identifies several active-site interactions important for the specific recognition of L-malate, but not other {alpha}-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-D-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.« less

Malate-mediated carbon catabolite repression in Bacillus subtilis involves the HPrK/CcpA pathway.

PubMed

Meyer, Frederik M; Jules, Matthieu; Mehne, Felix M P; Le Coq, Dominique; Landmann, Jens J; Görke, Boris; Aymerich, Stéphane; Stülke, Jörg

2011-12-01

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate.

Glycolysis without pyruvate kinase in Clostridium thermocellum

DOE PAGES

Olson, Daniel G.; Horl, Manuel; Fuhrer, Tobias; ...

2016-12-01

The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay.more » Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic 13C labeling. In the wild-type strain, the malate shunt accounts for about 33 ± 2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. Lastly, this provides the first direct evidence of the in-vivo function of the malate shunt.« less

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes.

PubMed

Singh, R; Chénier, D; Bériault, R; Mailloux, R; Hamel, R D; Appanna, V D

2005-09-30

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.

Determining In Vivo Regulation of Cardiac Pyruvate Dehydrogenase Based on Label Flux from Hyperpolarized [1-13C]Pyruvate

PubMed Central

Heather, Lisa C.; Griffin, Julian L.; Clarke, Kieran; Radda, George K.; Tyler, Damian J.

2015-01-01

Background Pyruvate dehydrogenase (PDH) is a key regulator of cardiac substrate selection and is regulated by both pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation and feedback inhibition. The extent to which chronic upregulation of PDK protein levels, acutely increased PDK activity and acute feedback inhibition limit PDH flux remains unclear because existing in vitro assessment methods inherently disrupt the enzyme complex. We have previously demonstrated that hyperpolarized 13C-labelled metabolic tracers with magnetic resonance spectroscopy (MRS) can monitor flux through PDH in vivo. The aim of this study was to determine the relative contributions of acute and chronic changes in PDK and PDH activities to in vivo myocardial PDH flux. Methodology/Principal Findings We examined both fed and fasted rats with either hyperpolarized [1-13C]pyruvate alone or hyperpolarized [1-13C]pyruvate co-infused with malate (to modulate mitochondrial NADH/NAD+ and acetyl-CoA/CoA ratios, which alter both PDH activity and flux). To confirm the metabolic fate of infused malate, we performed in vitro 1H NMR spectroscopy on cardiac tissue extracts. We observed that in fed rats, where PDH activity was high, the presence of malate increased PDH flux by 27%, whereas in the fasted state, malate infusion had no effect on PDH flux. Conclusions/Significance These observations suggest that pyruvate oxidation is limited by feedback inhibition from acetyl-CoA only when PDH activity is high. Therefore, in the case of PDH, and potentially other enzymes, hyperpolarized 13C MR can be used to non-invasively assess enzymatic regulation. PMID:21387444

Red Clover HCT2, a Hydroxycinnamoyl-Coenzyme A:Malate Hydroxycinnamoyl Transferase, Plays a Crucial Role in Biosynthesis of Phaselic Acid and Other Hydroxycinnamoyl-Malate Esters in Vivo1[OA

PubMed Central

Sullivan, Michael L.; Zarnowski, Robert

2011-01-01

In red clover (Trifolium pratense) leaves, phaselic acid (2-O-caffeoyl-l-malate) accumulates to several mmol kg−1 fresh weight and is a crucial component of a natural system that prevents protein breakdown during harvest and storage of this forage crop. Previously, we identified HCT2, a red clover gene encoding a hydroxycinnamoyl-Coenzyme A (CoA) hydroxycinnamoyl transferase capable of transferring p-coumaroyl and caffeoyl moieties from their CoA derivatives to malic acid to form the corresponding hydroxycinnamoyl-malate esters in vitro. Here, we carried out a detailed kinetic analysis of the enzyme and examined its in vivo function in red clover via reverse genetics. The kinetic analysis indicates that in vitro, despite similar Km values for the tested hydroxycinnamoyl-CoA derivatives, HCT2 favors transfer to malate of p-coumaroyl and feruloyl moieties over caffeoyl moieties by greater than 5-fold. Reverse reaction (transfer of hydroxycinnamoyl moieties from malate to CoA) by HCT2 was observed with p-coumaroyl-malate but not phaselic acid. Analysis of red clover plants down-regulated for HCT2 expression via RNA interference showed a significant and substantial correlation between HCT2 mRNA levels and phaselic acid accumulation (P < 0.005). In several of the HCT2-silenced plants, phaselic acid and p-coumaroyl-malate levels were reduced to <5% that of wild-type controls. These reductions resulted in easily observable phenotypes including reduced polyphenol oxidase-mediated browning and a reduction in blue epidermal fluorescence under ultraviolet light. These results demonstrate a crucial role for HCT2 in phaselic acid accumulation in red clover and define a previously undescribed pathway for the biosynthesis of hydroxycinnamoyl-malate esters in plants. PMID:21205620

Low pH, Aluminum, and Phosphorus Coordinately Regulate Malate Exudation through GmALMT1 to Improve Soybean Adaptation to Acid Soils1[W][OA

PubMed Central

Liang, Cuiyue; Piñeros, Miguel A.; Tian, Jiang; Yao, Zhufang; Sun, Lili; Liu, Jiping; Shaff, Jon; Coluccio, Alison; Kochian, Leon V.; Liao, Hong

2013-01-01

Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function. PMID:23341359

Low pH, aluminum, and phosphorus coordinately regulate malate exudation through GmALMT1 to improve soybean adaptation to acid soils.

PubMed

Liang, Cuiyue; Piñeros, Miguel A; Tian, Jiang; Yao, Zhufang; Sun, Lili; Liu, Jiping; Shaff, Jon; Coluccio, Alison; Kochian, Leon V; Liao, Hong

2013-03-01

Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function.

The R2R3-MYB transcription factor MdMYB73 is involved in malate accumulation and vacuolar acidification in apple.

PubMed

Hu, Da-Gang; Li, Yuan-Yuan; Zhang, Quan-Yan; Li, Ming; Sun, Cui-Hui; Yu, Jian-Qiang; Hao, Yu-Jin

2017-08-01

Malate, the predominant organic acid in many fruits, is a crucial component of the organoleptic quality of fruit, including taste and flavor. The genetic and environmental mechanisms affecting malate metabolism in fruit cells have been studied extensively. However, the transcriptional regulation of malate-metabolizing enzymes and vacuolar transporters remains poorly understood. Our previous studies demonstrated that MdMYB1 modulates anthocyanin accumulation and vacuolar acidification by directly activating vacuolar transporters, including MdVHA-B1, MdVHA-E, MdVHP1 and MdtDT. Interestingly, we isolated and identified a MYB transcription factor, MdMYB73, a distant relative of MdMYB1 in this study. It was subsequently found that MdMYB73 protein bound directly to the promoters of MdALMT9 (aluminum-activated malate transporter 9), MdVHA-A (vacuolar ATPase subunit A) and MdVHP1 (vacuolar pyrophosphatase 1), transcriptionally activating their expression and thereby enhancing their activities. Analyses of transgenic apple calli demonstrated that MdMYB73 influenced malate accumulation and vacuolar pH. Furthermore, MdCIbHLH1 interacted with MdMYB73 and enhanced its activity upon downstream target genes. These findings help to elucidate how MdMYB73 directly modulates the vacuolar transport system to affect malate accumulation and vacuolar pH in apple. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance

USDA-ARS?s Scientific Manuscript database

Aluminum (Al) activated root malate and citrate exudation play an important role in Al tolerance in many plant species. AtALMT1, an Al-activated malate transporter, is a major contributor to Arabidopsis Al tolerance. Here, we demonstrate that a second, unrelated gene, AtMATE, encodes an Arabidopsi...

Malate-Mediated Carbon Catabolite Repression in Bacillus subtilis Involves the HPrK/CcpA Pathway ▿ §

PubMed Central

Meyer, Frederik M.; Jules, Matthieu; Mehne, Felix M. P.; Le Coq, Dominique; Landmann, Jens J.; Görke, Boris; Aymerich, Stéphane; Stülke, Jörg

2011-01-01

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate. PMID:22001508

Oncogenic long noncoding RNA MALAT1 and HCV-related hepatocellular carcinoma.

PubMed

Toraih, Eman A; Ellawindy, Alia; Fala, Salma Y; Al Ageeli, Essam; Gouda, Nawal S; Fawzy, Manal S; Hosny, Somaya

2018-06-01

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. The oncogenic function of the long non-coding RNA; metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in HCC remains unclear. We aimed to evaluate MALAT1 serum expression profile in HCC and explore its relation to the clinicopathological features. Quantitative Real Time-Polymerase Chain Reaction was applied in 70 cohorts (30 HCC, 20 HCV, 20 controls). Further meta-analysis of clinical studies and in vitro validated experiments was employed. Serum MALAT1 showed area under the curve of 0.79 and 0.70 to distinguish patients with cancer from normal and cirrhotic individuals at fold change of 1.0 and 1.26, respectively. Expression level was significantly higher in males (P <0.001) and patients with massive ascites (P = 0.005). Correlation analysis showed positive correlation of MALAT1 with total bilirubin (r = 0.456, P <0.001) and AST (r = 0.280, P = 0.019), and negative correlation with the hemoglobin level (r = 0.312, P = 0.009). Meta-analysis showed that the over-expressed MALAT1 was linked to tumor number [Cohen's d = 0.450, 95% CI (0.21 to 0.68)], clinical stage [Cohen's d = 0.048, 95% CI (-0.83 to 0.74)], and AFP level [Cohen's d = 0.354, 95% CI (0.1 to 0.57)]. In silico data analysis and systematic review confirmed MALAT1 oncogenic function in cancer development and progression. In conclusion, circulatory MALAT1 might represent a putative non-invasive prognostic biomarker indicating worse liver failure score in HCV-related HCC patients with traditional markers. Large-scale verification is warranted in future studies. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

AtALMT9 is a malate-activated vacuolar chloride channel required for stomatal opening in Arabidopsis

PubMed Central

De Angeli, Alexis; Zhang, Jingbo; Meyer, Stefan; Martinoia, Enrico

2013-01-01

Water deficit strongly affects crop productivity. Plants control water loss and CO2 uptake by regulating the aperture of the stomatal pores within the leaf epidermis. Stomata aperture is regulated by the two guard cells forming the pore and changing their size in response to ion uptake and release. While our knowledge about potassium and chloride fluxes across the plasma membrane of guard cells is advanced, little is known about fluxes across the vacuolar membrane. Here we present the molecular identification of the long-sought-after vacuolar chloride channel. AtALMT9 is a chloride channel activated by physiological concentrations of cytosolic malate. Single-channel measurements demonstrate that this activation is due to a malate-dependent increase in the channel open probability. Arabidopsis thaliana atalmt9 knockout mutants exhibited impaired stomatal opening and wilt more slowly than the wild type. Our findings show that AtALMT9 is a vacuolar chloride channel having a major role in controlling stomata aperture. PMID:23653216

Identification and characterization of a class of MALAT1 -like genomic loci

DOE PAGES

Zhang, Bin; Mao, Yuntao S.; Diermeier, Sarah D.; ...

2017-05-23

The MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) gene encodes a noncoding RNA that is processed into a long nuclear retained transcript ( MALAT1) and a small cytoplasmic tRNA-like transcript (mascRNA). Using an RNA sequence- and structure-based covariance model, we identified more than 130 genomic loci in vertebrate genomes containing the MALAT1 3' end triple-helix structure and its immediate downstream tRNA-like structure, including 44 in the green lizard Anolis carolinensis. Structural and computational analyses revealed a co-occurrence of components of the 3' end module. MALAT1-like genes in Anolis carolinensis are highly expressed in adult testis, thus we named them testis-abundant longmore » noncoding RNAs (tancRNAs). MALAT1-like loci also produce multiple small RNA species, including PIWI-interacting RNAs (piRNAs), from the antisense strand. The 3' ends of tancRNAs serve as potential targets for the PIWI-piRNA complex. Furthermore, we have identified an evolutionarily conserved class of long noncoding RNAs (lncRNAs) with similar structural constraints, post-transcriptional processing, and subcellular localization and a distinct function in spermatocytes.« less

Identification and characterization of a class of MALAT1 -like genomic loci

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Bin; Mao, Yuntao S.; Diermeier, Sarah D.

The MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) gene encodes a noncoding RNA that is processed into a long nuclear retained transcript ( MALAT1) and a small cytoplasmic tRNA-like transcript (mascRNA). Using an RNA sequence- and structure-based covariance model, we identified more than 130 genomic loci in vertebrate genomes containing the MALAT1 3' end triple-helix structure and its immediate downstream tRNA-like structure, including 44 in the green lizard Anolis carolinensis. Structural and computational analyses revealed a co-occurrence of components of the 3' end module. MALAT1-like genes in Anolis carolinensis are highly expressed in adult testis, thus we named them testis-abundant longmore » noncoding RNAs (tancRNAs). MALAT1-like loci also produce multiple small RNA species, including PIWI-interacting RNAs (piRNAs), from the antisense strand. The 3' ends of tancRNAs serve as potential targets for the PIWI-piRNA complex. Furthermore, we have identified an evolutionarily conserved class of long noncoding RNAs (lncRNAs) with similar structural constraints, post-transcriptional processing, and subcellular localization and a distinct function in spermatocytes.« less

Molecular mechanisms of the non-coenzyme action of thiamin in brain: biochemical, structural and pathway analysis

PubMed Central

Mkrtchyan, Garik; Aleshin, Vasily; Parkhomenko, Yulia; Kaehne, Thilo; Luigi Di Salvo, Martino; Parroni, Alessia; Contestabile, Roberto; Vovk, Andrey; Bettendorff, Lucien; Bunik, Victoria

2015-01-01

Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDP-dependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins. PMID:26212886

Temperature enhanced succinate production concurrent with increased central metabolism turnover in the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Hasunuma, Tomohisa; Matsuda, Mami; Kato, Yuichi; Vavricka, Christopher John; Kondo, Akihiko

2018-05-27

Succinate is a versatile petrochemical compound that can be produced by microorganisms, often from carbohydrate based carbon sources. Phototrophic cyanobacteria including Synechocystis sp. PCC 6803 can more efficiently produce organic acids such as succinate without sugar supplementation, via photosynthetic production of glycogen followed by glycogen utilization, typically under dark conditions. In this study, Synechocystis 6803 bioproduction of organic acids under dark anoxic conditions was found to increase with elevation of temperature from 30 °C to 37 °C. The further enhancement of succinate bioproduction by overexpression of the rate limiting enzyme phosphoenolpyruvate carboxylase resulted in improved glycogen utilization. To gain more insight into the mechanisms underlying the increased organic acid output, a novel temperature dependent metabolomics analysis was performed. Adenylate energy charge was found to decrease along with elevating temperature, while central metabolites glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glycerol 3-phosphate, malate, fumarate and succinate increased. Temperature dependent 13 C-labeling metabolomics analysis further revealed a glycolysis to TCA bottleneck, which could be overcome by addition of CO 2 , leading to even higher organic acid production. Optimization of initial cell concentration to 25 g-dry cell weight/L, in combination with 100 mM NaHCO 3 supplementation, afforded a succinate titer of over 1.8 g/L, the highest reported autotrophic succinate titer. Succinate titers remained high after additional knockout of ackA, resulting in the highest reported autotrophic D-lactate titer as well. The optimization of Synechocystis 6803 organic acid production therefore holds significant promise for CO 2 capture and utilization. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Spectral Reflectance Properties of Wetlands Plants

DTIC Science & Technology

1994-08-01

metabolism (Figure 1). Flood-intolerant plants also produce malate ; however, malate is converted to pyruvate, which is further reduced to ethanol...adapted to flooded conditions. For example, flood tolerance has been linked to the production and accumulation of non-toxic malate as a by- product of...oxidation (REDOX) potential. REDOX potential Figure l. Metabolic pathways for o tolernt and flood tolernt describes the reducing plants dted from MiLc

17β-Estradiol regulates cell proliferation, colony formation, migration, invasion and promotes apoptosis by upregulating miR-9 and thus degrades MALAT-1 in osteosarcoma cell MG-63 in an estrogen receptor-independent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Dengfeng; Yang, Hui; Lin, Jing

2015-02-20

In bone, different concentration of estrogen leads to various of physiological processes in osteoblast, such as the proliferation, migration, and apoptosis in an estrogen receptor-dependent manner. But little was known about the estrogen effects on osteosarcoma (OS). In this study, OS cell MG-63 was treated with low (1 nM) or high (100 nM) dose of 17β-Estradiol (E2) with the presence or absence of estrogen receptor α (ERα), for evaluating the E2 effects on proliferation, migration, invasion, colony formation and apoptosis. Consistent with a previous study, high dose of E2 treatment dramatically downregulated expressing level of long non-coding RNA metastasis associated lung adenocarcinomamore » transcript 1 (MALAT-1). The observation of upregulation of miR-9 after a high dose of E2 treatment indicated the cause of MALAT-1 reduction. Downregulation of MALAT-1 promoted the combination of SFPQ/PTBP2 complex. It was also observed that the proliferation, migration, invasion, colony formation and apoptosis of OS cells were remarkably affected by high dose of E2 treatment, but not by low dose, in an ERα independent manner. Furthermore, the abolishment of the effects on these physiological processes caused by ectopic expression of miR-9 ASOs suggested the necessity of miR-9 in MALAT-1 regulation. Here we found that the high dose of E2 treatment upregulated miR-9 thus posttranscriptionally regulated MALAT-1 RNA level in OS cells, and then the downregulation of MALAT-1 inhibited cell proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) processes in the E2-dose dependent and ER-independent ways. - Highlights: • E2 affects osteosarcoma cell MG-63 in an Estrogen receptor-independent way. • High dose of E2 treatment upregulates miR-9 which target to MALAT-1 RNA. • Upregulated miR-9 degrades MALAT-1 and thus affects combination of SFPQ/PTBP2. • E2 treatment block cell proliferation, colony formation, mobility, and enhance apoptosis.« less

Critical role of mitochondrial ROS is dependent on their site of production on the electron transport chain in ischemic heart.

PubMed

Madungwe, Ngonidzashe B; Zilberstein, Netanel F; Feng, Yansheng; Bopassa, Jean C

2016-01-01

Reactive oxygen species (ROS) generation has been implicated in many pathologies including ischemia/reperfusion (I/R) injury. This led to multiple studies on antioxidant therapies to treat cardiovascular diseases but paradoxically, results have so far been mixed as ROS production can be beneficial as a signaling mechanism and in cardiac protection via preconditioning interventions. We investigated whether the differential impact of increased ROS in injury as well as in protection could be explained by their site of production on the mitochondrial electron transport chain. Using amplex red to measure ROS production, we found that mitochondria isolated from hearts after I/R produced more ROS than non-ischemic when complex I substrate (glutamate/malate) was used. Interestingly, the substrates of complex II (succinate) and ubiquinone (sn-glycerol 3-phosphate, G3P) produced less ROS in mitochondria from I/R hearts compared to normal healthy hearts. The inhibitors of complex I (rotenone) and complex III (antimycin A) increased ROS production when glutamate/malate and G3P were used; in contrast, they reduced ROS production when the complex II substrate was used. Mitochondrial calcium retention capacity required to induce mitochondrial permeability transition pore (mPTP) opening was measured using calcium green fluorescence and was found to be higher when mitochondria were treated with G3P and succinate compared to glutamate/malate. Furthermore, Langendorff hearts treated with glutamate/malate exhibited reduced cardiac functional recovery and increased myocardial infarct size compared to hearts treated with G3P. Thus, ROS production by the stimulated respiratory chain complexes I and III has opposite roles: cardio-deleterious when produced in complex I and cardio-protective when produced in complex III. The mechanism of these ROS involves the inhibition of the mPTP opening, a key event in cell death following ischemia/reperfusion injury.

Ammonium intensifies CAM photosynthesis and counteracts drought effects by increasing malate transport and antioxidant capacity in Guzmania monostachia.

PubMed

Pereira, Paula Natália; Gaspar, Marília; Smith, J Andrew C; Mercier, Helenice

2018-04-09

Guzmania monostachia (Bromeliaceae) is a tropical epiphyte capable of up-regulating crassulacean acid metabolism (CAM) in its photosynthetic tissues in response to changing nutrient and water availability. Previous studies have shown that under drought there is a gradient of increasing CAM expression from the basal (youngest) to the apical (oldest) portion of the leaves, and additionally that nitrogen deficiency can further increase CAM intensity in the leaf apex of this bromeliad. The present study investigated the inter-relationships between nitrogen source (nitrate and/or ammonium) and water deficit in regulating CAM expression in G. monostachia leaves. The highest CAM activity was observed under ammonium nutrition in combination with water deficit. This was associated with enhanced activity of the key enzyme phosphoenolpyruvate carboxylase, elevated rates of ATP- and PPi-dependent proton transport at the vacuolar membrane in the presence of malate, and increased transcript levels of the vacuolar malate channel-encoding gene, ALMT. Water deficit was consistently associated with higher levels of total soluble sugars, which were maximal under ammonium nutrition, as were the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase). Thus, ammonium nutrition, whilst associated with the highest degree of CAM induction in G. monostachia, also mitigates the effects of water deficit by osmotic adjustment and can limit oxidative damage in the leaves of this bromeliad under conditions that may be typical of its epiphytic habitat.

Microbial biosynthesis and secretion of l-malic acid and its applications.

PubMed

Chi, Zhe; Wang, Zhi-Peng; Wang, Guang-Yuan; Khan, Ibrar; Chi, Zhen-Ming

2016-01-01

l-Malic acid has many uses in food, beverage, pharmaceutical, chemical and medical industries. It can be produced by one-step fermentation, enzymatic transformation of fumaric acid to l-malate and acid hydrolysis of polymalic acid. However, the process for one-step fermentation is preferred as it has many advantages over any other process. The pathways of l-malic acid biosynthesis in microorganisms are partially clear and three metabolic pathways including non-oxidative pathway, oxidative pathway and glyoxylate cycle for the production of l-malic acid from glucose have been identified. Usually, high levels of l-malate are produced under the nitrogen starvation conditions, l-malate, as a calcium salt, is secreted from microbial cells and CaCO3 can play an important role in calcium malate biosynthesis and regulation. However, it is still unclear how it is secreted into the medium. To enhance l-malate biosynthesis and secretion by microbial cells, it is very important to study the mechanisms of l-malic acid biosynthesis and secretion at enzymatic and molecular levels.

Mutation and Selection of Lactobacillus plantarum Strains That Do Not Produce Carbon Dioxide from Malate †

PubMed Central

Daeschel, M. A.; McFeeters, R. F.; Fleming, H. P.; Klaenhammer, T. R.; Sanozky, R. B.

1984-01-01

A differential medium was developed to distinguish between malate-decarboxylating (MDC+) and -non-decarboxylating (MDC−) strains of Lactobacillus plantarum. MDC− strains produced a visible acid reaction in the medium, whereas MDC+ strains did not. Use of the medium allowed for rapid screening and isolation of mutagenized cells that had lost the ability to produce CO2 from malate. PMID:16346479

Malate Plays a Crucial Role in Starch Metabolism, Ripening, and Soluble Solid Content of Tomato Fruit and Affects Postharvest Softening[W][OA

PubMed Central

Centeno, Danilo C.; Osorio, Sonia; Nunes-Nesi, Adriano; Bertolo, Ana L.F.; Carneiro, Raphael T.; Araújo, Wagner L.; Steinhauser, Marie-Caroline; Michalska, Justyna; Rohrmann, Johannes; Geigenberger, Peter; Oliver, Sandra N.; Stitt, Mark; Carrari, Fernando; Rose, Jocelyn K.C.; Fernie, Alisdair R.

2011-01-01

Despite the fact that the organic acid content of a fruit is regarded as one of its most commercially important quality traits when assessed by the consumer, relatively little is known concerning the physiological importance of organic acid metabolism for the fruit itself. Here, we evaluate the effect of modifying malate metabolism in a fruit-specific manner, by reduction of the activities of either mitochondrial malate dehydrogenase or fumarase, via targeted antisense approaches in tomato (Solanum lycopersicum). While these genetic perturbations had relatively little effect on the total fruit yield, they had dramatic consequences for fruit metabolism, as well as unanticipated changes in postharvest shelf life and susceptibility to bacterial infection. Detailed characterization suggested that the rate of ripening was essentially unaltered but that lines containing higher malate were characterized by lower levels of transitory starch and a lower soluble sugars content at harvest, whereas those with lower malate contained higher levels of these carbohydrates. Analysis of the activation state of ADP-glucose pyrophosphorylase revealed that it correlated with the accumulation of transitory starch. Taken together with the altered activation state of the plastidial malate dehydrogenase and the modified pigment biosynthesis of the transgenic lines, these results suggest that the phenotypes are due to an altered cellular redox status. The combined data reveal the importance of malate metabolism in tomato fruit metabolism and development and confirm the importance of transitory starch in the determination of agronomic yield in this species. PMID:21239646

Glutamine and ornithine alpha-ketoglutarate supplementation on malate dehydrogenases expression in hepatectomized rats.

PubMed

Guimarães Filho, Artur; Cunha, Rodrigo Maranguape Silva da; Vasconcelos, Paulo Roberto Leitão de; Guimarães, Sergio Botelho

2014-06-01

To evaluate the relative gene expression (RGE) of cytosolic (MDH1) and mitochondrial (MDH2) malate dehydrogenases enzymes in partially hepatectomized rats after glutamine (GLN) or ornithine alpha-ketoglutarate (OKG) suplementation. One-hundred and eight male Wistar rats were randomly distributed into six groups (n=18): CCaL, GLNL and OKGL and fed calcium caseinate (CCa), GLN and OKG, 0.5 g/Kg by gavage, 30 minutes before laparotomy. CCaH, GLNH and OKGH groups were likewise fed 30 minutes before 70% partial hepatectomy. Blood and liver samples were collected three, seven and 14 days after laparotomy/hepatectomy for quantification of MDH1/MDH2 enzymes using the real-time polymerase chain reaction (PCR) methodology. Relative enzymes expression was calculated by the 2-(ΔΔC)T method using the threshold cycle (CT) value for normalization. MDH1/MDH2 RGE was not different in hepatectomized rats treated with OKG compared to rats treated with CCa. However, MDH1/MDH2 RGE was greater on days 3 (321:1/26.48:1) and 7 (2.12:1/2.48:1) while MDH2 RGE was greater on day 14 (7.79:1) in hepatectomized rats treated with GLN compared to control animals. Glutamine has beneficial effects in liver regeneration in rats by promoting an up-regulation of the MDH1 and MDH2 relative gene expression.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grissom, C.B.; Cleland, W.W.

The role of the metal ion in the oxidative decarboxylation of malate by chicken liver NADP malic enzyme and details of the reaction mechanism have been investigated by /sup 13/C isotope effects. With saturating NADP and the indicated metal ion at a total concentration 10-fold higher than its K/sub m/, the following primary /sup 13/C kinetic isotope effects at C/sub 4/ of malate (/sup 13/(VK/sub mal/)) were observed at pH 8.0: Mg/sup 2 +/, 1.0336; Mn/sup 2 +/, 1.0365; Cd/sup 2 +/, 1.0366; Zn/sup 2 +/, 1.0337; Co/sup 2 +/, 1.0283; Ni/sup 2 +/, 1.025. Knowing the partitioning of themore » intermediate oxalacetate between decarboxylation to pyuvate and reduction to malate allows calculation of the intrinsic carbon isotope effect for decarboxylation to pyuvate and reduction to malate allows calculation of the intrinsic carbon isotope effect for decarboxylation. For Mg/sup 2 +/ as activator, this was 1.049 with NADP and 1.046 with 3-acetylpyridine adenine dinucleotide phosphate, although the intrinsic primary deuterium isotope effects on dehydrogenation were 5.6 and 4.2, and the partition ratios of the oxalacetate intermediate for decarboxylation as opposed to hydride transfer were 0.11 and 3.96. It was not possible to calculate reasonable intrinsic carbon isotope effects with the other metal ions by use of the partitioning ratio of oxalacetate because of decarboxylation by another mechanism. The variation of /sup 13/(VK/sub mal/) with pH was used to dissect the total forward and external components. When the authors attempted to use the variation of /sup 13/(VK/sub mal/) with solution viscosity to determine the internal and external commitments, incorrect values were obtained because of a specific effect of the viscosogen in decreasing the K/sub m/ for malate, so that VK/sub mal/ actually increased with viscosity instead of decreasing, as theory predicts.« less

Malate valves: Old shuttles with new perspectives.

PubMed

Selinski, Jennifer; Scheibe, Renate

2018-06-22

Malate valves act as powerful systems for balancing the ATP/NAD(P)H ratio required in various subcellular compartments in plant cells. As components of malate valves, isoforms of malate dehydrogenases (MDHs) and dicarboxylate translocators catalyze the reversible interconversion of malate and oxaloacetate and their transport. Depending on the coenzyme specificity of the MDH isoforms, either NADH or NADPH can be transported indirectly. Arabidopsis thaliana possesses nine genes encoding MDH isoenzymes: Activities of NAD-dependent MDHs have been detected in mitochondria, peroxisomes, cytosol and plastids, respectively. In addition, chloroplasts possess a NADP-dependent MDH isoform. The NADP-MDH as part of the "light malate valve" plays an important role as a poising mechanism to adjust the ATP/NADPH ratio in the stroma. Its activity is strictly regulated by post-translational redox-modification mediated via the ferredoxin-thioredoxin system and fine control via the NADP + /NADP(H) ratio, thereby maintaining redox homeostasis under changing conditions. In contrast, the plastid NAD-MDH ("dark malate valve") is constitutively active and its lack leads to failure in early embryo development. While redox regulation of the main cytosolic MDH isoform has been shown, the knowledge about regulation of the other two cytosolic MDHs as well as NAD-MDH isoforms from peroxisomes and mitochondria is still lacking. Knockout mutants lacking the isoforms from chloroplasts, mitochondria, and peroxisomes have been characterized, but not much is known about cytosolic NAD-MDH isoforms and their role in planta. This review updates the current knowledge on MDH isoforms and the shuttle systems for intercompartmental dicarboxylate exchange focusing on the various metabolic functions of these valves. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Characterization of mitochondrial dicarboxylate/tricarboxylate transporters from grape berries.

PubMed

Regalado, Ana; Pierri, Ciro Leonardo; Bitetto, Maria; Laera, Valentina Liliana; Pimentel, Catarina; Francisco, Rita; Passarinho, José; Chaves, Maria M; Agrimi, Gennaro

2013-03-01

Grape berries (Vitis vinifera L fruit) exhibit a double-sigmoid pattern of development that results from two successive periods of vacuolar swelling during which the nature of accumulated solutes changes significantly. Throughout the first period, called green or herbaceous stage, berries accumulate high levels of organic acids, mainly malate and tartrate. At the cellular level fruit acidity comprises both metabolism and vacuolar storage. Malic acid compartmentation is critical for optimal functioning of cytosolic enzymes. Therefore, the identification and characterization of the carriers involved in malate transport across sub-cellular compartments is of great importance. The decrease in acid content during grape berry ripening has been mainly associated to mitochondrial malate oxidation. However, no Vitis vinifera mitochondrial carrier involved in malate transport has been reported to date. Here we describe the identification of three V. vinifera mitochondrial dicarboxylate/tricarboxylate carriers (VvDTC1-3) putatively involved in mitochondrial malate, citrate and other di/tricarboxylates transport. The three VvDTCs are very similar, sharing a percentage of identical residues of at least 83 %. Expression analysis of the encoding VvDTC genes in grape berries shows that they are differentially regulated exhibiting a developmental pattern of expression. The simultaneous high expression of both VvDTC2 and VvDTC3 in grape berry mesocarp close to the onset of ripening suggests that these carriers might be involved in the transport of malate into mitochondria.

A chimeric protein of aluminum-activated malate transporter generated from wheat and Arabidopsis shows enhanced response to trivalent cations.

PubMed

Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Yamamoto, Yoko

2016-07-01

TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Prognostic value of long noncoding RNA MALAT1 in digestive system malignancies.

PubMed

Zhai, Hui; Li, Xiao-Mei; Maimaiti, Ailifeire; Chen, Qing-Jie; Liao, Wu; Lai, Hong-Mei; Liu, Fen; Yang, Yi-Ning

2015-01-01

MALAT1, a newly discovered long noncoding RNA (lncRNA), has been reported to be highly expressed in many types of cancers. This meta-analysis summarizes its potential prognostic value in digestive system malignancies. A quantitative meta-analysis was performed through a systematic search in PubMed, Cochrane Library, Web of Science and Chinese National Knowledge Infrastructure (CNKI) for eligible papers on the prognostic impact of MALAT1 in digestive system malignancies from inception to Apr. 25, 2015. Pooled hazard ratios (HRs) with 95% confidence interval (95% CI) were calculated to summarize the effect. Five studies were included in the study, with a total of 527 patients. A significant association was observed between MALAT1 abundance and poor overall survival (OS) of patients with digestive system malignancies, with pooled hazard ratio (HR) of 7.68 (95% confidence interval [CI]: 4.32-13.66, P<0.001). Meta sensitivity analysis suggested the reliability of our findings. No publication bias was observed. MALAT1 abundance may serve as a novel predictive factor for poor prognosis in patients with digestive system malignancies.

Prognostic value of long noncoding RNA MALAT1 in digestive system malignancies

PubMed Central

Zhai, Hui; Li, Xiao-Mei; Maimaiti, Ailifeire; Chen, Qing-Jie; Liao, Wu; Lai, Hong-Mei; Liu, Fen; Yang, Yi-Ning

2015-01-01

Background: MALAT1, a newly discovered long noncoding RNA (lncRNA), has been reported to be highly expressed in many types of cancers. This meta-analysis summarizes its potential prognostic value in digestive system malignancies. Methods: A quantitative meta-analysis was performed through a systematic search in PubMed, Cochrane Library, Web of Science and Chinese National Knowledge Infrastructure (CNKI) for eligible papers on the prognostic impact of MALAT1 in digestive system malignancies from inception to Apr. 25, 2015. Pooled hazard ratios (HRs) with 95% confidence interval (95% CI) were calculated to summarize the effect. Results: Five studies were included in the study, with a total of 527 patients. A significant association was observed between MALAT1 abundance and poor overall survival (OS) of patients with digestive system malignancies, with pooled hazard ratio (HR) of 7.68 (95% confidence interval [CI]: 4.32-13.66, P<0.001). Meta sensitivity analysis suggested the reliability of our findings. No publication bias was observed. Conclusions: MALAT1 abundance may serve as a novel predictive factor for poor prognosis in patients with digestive system malignancies. PMID:26770406

Cloning and characterization of pyruvate carboxylase gene responsible for calcium malate overproduction in Penicillium viticola 152 and its expression analysis.

PubMed

Khan, Ibrar; Qayyum, Sadia; Ahmed, Shehzad; Maqbool, Farhana; Tauseef, Isfahan; Haleem, Kashif Syed; Chi, Zhen-Ming

2017-03-20

In this study, a pyruvate carboxylase gene (PYC) from a marine fungus Penicillium viticola 152 isolated from marine algae was cloned and characterized by using Genome Walking method. An open reading frame (ORF) of The PYC gene (accession number: KM593097) had 3582bp encoding 1193 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 131.2757kDa. A putative promoter (intronless) of the gene was located at -666bp and contained a TATA box, several CAAT boxes, the 5'-SYGGRG-3' and a 5'-HGATAR-3' sequences. A consensus polyadenylation site (AATAAA) was also observed at +10bp downstream of the ORF. The protein deduced from the PYC gene had no signal peptide, was a homotetramer (4), and had the four functional domains. Furthermore, PYC protein also had three potential N-linked glycosylation sites, among them, -N-S-T-I- at 36 amino acid, -N-G-T-V- at 237 amino acid, and -N-G-S-S- at 517 amino acid were the most possible N-glycosylation sites. After expression of the PYC gene of P. viticola 152 in medium supplemented with CSL and biotin, it was found that the specific pyruvate carboxylase activity in MA production medium supplemented with CSL was much higher (0.5U/mg) than in MA medium supplemented with biotin (0.3U/mg), suggesting that optimal concentration of CSL is required for increased expression of the PYC gene, which is responsible for high level production of malic acid in P. viticola 152 strain. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of granulation on organic acid metabolism and its relation to mineral elements in Citrus grandis juice sacs.

PubMed

Wang, Xian-You; Wang, Ping; Qi, Yi-Ping; Zhou, Chen-Ping; Yang, Lin-Tong; Liao, Xin-Yan; Wang, Liu-Qing; Zhu, Dong-Huang; Chen, Li-Song

2014-02-15

We investigated the effects of granulation on organic acid metabolism and its relation to mineral elements in 'Guanximiyou' pummelo (Citrus grandis) juice sacs. Granulated juice sacs had decreased concentrations of citrate and isocitrate, thus lowering juice sac acidity. By contrast, malate concentration was higher in granulated juice sacs than in normal ones. The reduction in citrate concentration might be caused by increased degradation, as indicated by enhanced aconitase activity, whilst the increase in malate concentration might be caused by increased biosynthesis, as indicated by enhanced phosphoenolpyruvate carboxylase (PEPC). Real time quantitative reverse transcription PCR (qRT-PCR) analysis showed that the activities of most acid-metabolizing enzymes were regulated at the transcriptional level, whilst post-translational modifications might influence the PEPC activity. Granulation led to increased accumulation of mineral elements (especially phosphorus, magnesium, sulphur, zinc and copper) in juice sacs, which might be involved in the incidence of granulation in pummelo fruits. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification and Functional Characterization of a Tonoplast Dicarboxylate Transporter in Tomato (Solanum lycopersicum)

PubMed Central

Liu, Ruiling; Li, Boqiang; Qin, Guozheng; Zhang, Zhanquan; Tian, Shiping

2017-01-01

Acidity plays an important role in flavor and overall organoleptic quality of fruit and is mainly due to the presence of organic acids. Understanding the molecular basis of organic acid metabolism is thus of primary importance for fruit quality improvement. Here, we cloned a putative tonoplast dicarboxylate transporter gene (SlTDT) from tomato, and submitted it to the NCBI database (GenBank accession number: KC733165). SlTDT protein contained 13 putative transmembrane domains in silico analysis. Confocal microscopic study using green fluorescent fusion proteins revealed that SlTDT was localized on tonoplast. The expression patterns of SlTDT in tomato were analyzed by RT-qPCR. The results indicated that SlTDT expressed in leaves, roots, flowers and fruits at different ripening stages, suggesting SlTDT may be associated with the development of different tissues. To further explore the function of SlTDT, we constructed both overexpression and RNAi vectors and obtained transgenic tomato plants by agrobacterium-mediated method. Gas chromatography-mass spectrometer (GC-MS) analysis showed that overexpression of SlTDT significantly increased malate content, and reduced citrate content in tomato fruit. By contrast, repression of SlTDT in tomato reduced malate content of and increased citrate content. These results indicated that SlTDT played an important role in remobilization of malate and citrate in fruit vacuoles. PMID:28261242

Identification and Functional Characterization of a Tonoplast Dicarboxylate Transporter in Tomato (Solanum lycopersicum).

PubMed

Liu, Ruiling; Li, Boqiang; Qin, Guozheng; Zhang, Zhanquan; Tian, Shiping

2017-01-01

Acidity plays an important role in flavor and overall organoleptic quality of fruit and is mainly due to the presence of organic acids. Understanding the molecular basis of organic acid metabolism is thus of primary importance for fruit quality improvement. Here, we cloned a putative tonoplast dicarboxylate transporter gene ( SlTDT ) from tomato, and submitted it to the NCBI database (GenBank accession number: KC733165). SlTDT protein contained 13 putative transmembrane domains in silico analysis. Confocal microscopic study using green fluorescent fusion proteins revealed that SlTDT was localized on tonoplast. The expression patterns of SlTDT in tomato were analyzed by RT-qPCR. The results indicated that SlTDT expressed in leaves, roots, flowers and fruits at different ripening stages, suggesting SlTDT may be associated with the development of different tissues. To further explore the function of SlTDT , we constructed both overexpression and RNAi vectors and obtained transgenic tomato plants by agrobacterium-mediated method. Gas chromatography-mass spectrometer (GC-MS) analysis showed that overexpression of SlTDT significantly increased malate content, and reduced citrate content in tomato fruit. By contrast, repression of SlTDT in tomato reduced malate content of and increased citrate content. These results indicated that SlTDT played an important role in remobilization of malate and citrate in fruit vacuoles.

Adaptation to acidic soil is achieved by increased numbers of cis-acting elements regulating ALMT1 expression in Holcus lanatus.

PubMed

Chen, Zhi Chang; Yokosho, Kengo; Kashino, Miho; Zhao, Fang-Jie; Yamaji, Naoki; Ma, Jian Feng

2013-10-01

Yorkshire fog (Holcus lanatus), which belongs to the Poaceae family and is a close relative of the agronomic crop oat (Avena sativa), is a widely adaptable grass species that is able to grow on highly acidic soils with high levels of Al, but the mechanism underlying the high Al tolerance is unknown. Here, we characterized two accessions of H. lanatus collected from an acid plot (soil pH 3.6, HL-A) and a neutral plot (pH 7.1, HL-N) in terms of Al tolerance, organic acid anion secretion and related gene expression. In response to Al (pH 4.5), the HL-A roots secreted approximately twice as much malate as the HL-N roots, but there was no difference in citrate secretion. Cloning of the gene HlALMT1 responsible for malate secretion showed that the encoded amino acid sequence did not differ between two accessions, but the expression level in the outer cell layers of the HL-A roots was twice as high as in the HL-N roots. This difference was not due to the genomic copy number, but was due to the number of cis-acting elements for an Al-responsive transcription factor (HlART1) in the promoter region of HlALMT1, as demonstrated by both a yeast one-hybrid assay and a transient assay in tobacco protoplasts. Furthermore, introduction of HlALMT1 driven by the HL-A promoter into rice resulted in significantly more Al-induced malate secretion than introduction of HlALMT1 driven by the HL-N promoter. These findings indicate that the adaptation of H. lanatus to acidic soils may be achieved by increasing number of cis-acting elements for ART1 in the promoter region of the HlALMT1 gene, enhancing the expression of HlALMT1 and the secretion of malate. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Effects of microgravity and tail suspension on enzymes of individual soleus and tibialis anterior fibers

NASA Technical Reports Server (NTRS)

Chi, Maggie M.-Y.; Choski, Rati; Nemeth, Patti; Krasnov, Igor'; Il'ina-Kakueva, E. I.; Manchester, Jill K.; Lowry, Oliver H.

1992-01-01

Selected enzymes of energy metabolism were measured in random individual fibers of soleus and tibialis anterior (TA) muscles from rats exposed for 2 wk to spaceflight (F) aboard Cosmos 2044 or tail suspension (T) and from synchronous controls. Average size of soleus fibers (dry weight per unit length) was reduced 37 percent in F and T fibers; there was little change in Ta fibers. Enzyme changes were more pronounced in soleus than in TA fibers. Three enzymes characteristic of fast-twitch muscles, pyruvate kinase, glycerol-3-phosphate dehydrogenase, and 1-phosphofructokinase, were elevated in F and T soleus fibers, but changes in phosphofructokinase were not statistically significant. In TA fibers analyzed for hexokinase, malate dehydrogenase, phosphohexoisomerase, and pyruvate kinase, only hexokinase and malate dehydrogenase showed significant changes. Hexokinase incresed 83 percent in one of two T muscles. Enzyme data for TA fibers typed by myosin adenosinetriphosphatase were more informative: phosphofructokinase, phosphorylase, and glycerol-3-phosphate dehydrogenase were increased in type IIn fibers of either F or T muscles or both. Malate dehydrogenase was not changed in fibers of any type in either F or T muscle.

Systemic Sunitinib Malate Treatment for Advanced Juxtapapillary Retinal Hemangioblastomas Associated with von Hippel-Lindau Disease.

PubMed

Knickelbein, Jared E; Jacobs-El, Naima; Wong, Wai T; Wiley, Henry E; Cukras, Catherine A; Meyerle, Catherine B; Chew, Emily Y

2017-01-01

To describe the clinical course of advanced juxtapapillary retinal capillary hemangioblastomas (RCH) associated with von Hippel-Lindau (VHL) disease treated with systemic sunitinib malate, an agent that inhibits both anti-vascular endothelial growth factor and anti-platelet-derived growth factor signaling. Observational case review. Three patients with advanced VHL-related juxtapapillary RCH treated with systemic sunitinib malate. Patient 1 was followed routinely every 4 months while on systemic sunitinib prescribed by her oncologist for metastatic pancreatic neuroendocrine and kidney tumors. Patients 2 and 3 were part of a prospective clinical trial evaluating the use of systemic sunitinib for ocular VHL lesions during a period of 9 months. Visual acuity, size of RCH, and degree of exudation were recorded at each visit. Optical coherence tomography (OCT) and fluorescein angiography were also obtained at some visits. Visual acuity, size of RCH, and degree of exudation. Three patients with advanced VHL-associated juxtapapillary RCH were treated with systemic sunitinib malate. While none of the patients lost vision during therapy, treatment with sunitinib malate did not improve visual acuity or reduce the size of RCH. Improvements in RCH-associated retinal edema were observed in two patients. All patients experienced multiple adverse effects, including thyroid toxicity, thrombocytopenia, nausea, fatigue, jaundice, and muscle aches. Two of the three patients had to discontinue treatment prematurely and the third required dose reduction. Systemic sunitinib malate may be useful in slowing progression of ocular disease from VHL-associated RCH. However, significant systemic adverse effects limited its use in this small series, and systemic sunitinib malate may not be safe for treatment of RCH when used at the doses described in this report. Further studies are required to determine if this medication used at lower doses with different treatment strategies, other medications in the same class or drugs directed at multiple targets in the tumor, may be safer and more effective for the treatment of advanced VHL-associated RCH.

Effects of Al(III) and Nano-Al13 Species on Malate Dehydrogenase Activity

PubMed Central

Yang, Xiaodi; Cai, Ling; Peng, Yu; Li, Huihui; Chen, Rong Fu; Shen, Ren Fang

2011-01-01

The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al13 can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al13 concentration increase. Our study also found that the effects of Al(III) and Al13 on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules. PMID:22163924

Effects of Al(III) and nano-Al13 species on malate dehydrogenase activity.

PubMed

Yang, Xiaodi; Cai, Ling; Peng, Yu; Li, Huihui; Chen, Rong Fu; Shen, Ren Fang

2011-01-01

The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al(13) can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al(13) concentration increase. Our study also found that the effects of Al(III) and Al(13) on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules.

[Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

PubMed

Voloshchuk, O N; Kopylchuk, G P

2016-01-01

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage.

Clinical prognostic value of metastasis-associated lung adenocarcinoma transcript 1 in various human cancers: an updated meta-analysis.

PubMed

Li, Yang; Yang, Ze; Wan, Xiaoya; Zhou, Jianguo; Zhang, Yu; Ma, Hu; Bai, Yuju

2016-05-28

Many studies have investigated the prognostic value of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in human cancers. However, these studies were often limited by small sample sizes. Therefore, we performed this updated meta-analysis to summarize the potential value of MALAT1 as a biomarker for early treatment and to predict survival in various human malignant neoplasms, through the inclusion of the latest literature and improved methodology. Twelve eligible articles were systematically obtained from PubMed, Medline, Embase, Web of Science, China National Knowledge Infrastructure and the Cochrane Library, from inception up to June 30, 2015. Survival was assessed using pooled hazard ratios (HRs) and 95% confidence intervals (95% CIs). By combining the results of 12 studies, we found elevated MALAT1 expression was associated with poor survival in most cancers, with a pooled HR of 1.90 (95% CI, 1.56-2.30) for overall survival (OS) and 3.06 (95% CI, 2.06-4.56) for recurrence-free survival/disease-free survival. Subgroup analyses according to ethnicity, tumor type, assay method, sample size, HR-calculation method and analysis type did not affect the predictive role of MALAT1 for OS in various cancer types. Further, by combining results from studies that used multivariate analyses, we found elevated MALAT1 was an independent prognostic factor for OS (HR = 1.98; 95% CI, 1.58-2.48). MALAT1 could serve as a potential prognostic biomarker in various cancers and may be a potential therapeutic target for the treatment and early detection of recurrence.

Myostatin-induced inhibition of the long noncoding RNA Malat1 is associated with decreased myogenesis.

PubMed

Watts, Rani; Johnsen, Virginia L; Shearer, Jane; Hittel, Dustin S

2013-05-15

Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily of secreted proteins, is a potent negative regulator of myogenesis. Free myostatin induces the phosphorylation of the Smad family of transcription factors, which, in turn, regulates gene expression, via the canonical TGF-β signaling pathway. There is, however, emerging evidence that myostatin can regulate gene expression independent of Smad signaling. As such, we acquired global gene expression data from the gastrocnemius muscle of C57BL/6 mice following a 6-day treatment with recombinant myostatin compared with vehicle-treated animals. Of the many differentially expressed genes, the myostatin-associated decrease (-11.20-fold; P < 0.05) in the noncoding metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was the most significant and the most intriguing because of numerous reports describing its novel role in regulating cell growth. We therefore sought to further characterize the role of Malat1 expression in skeletal muscle myogenesis. RT-PCR-based quantification of C2C12 and primary human skeletal muscle cells revealed a significant and persistent upregulation (4- to 7-fold; P < 0.05) of Malat1 mRNA during the differentiation of myoblasts into myotubes. Conversely, targeted knockdown of Malat1 using siRNA suppressed myoblast proliferation by arresting cell growth in the G(0)/G(1) phase. These results reveal Malat1 as novel downstream target of myostatin with a considerable ability to regulate myogenesis. The identification of new targets of myostatin will have important repercussions for regenerative biology through inhibition and/or reversal of muscle atrophy and wasting diseases.

The importance of nodule CO2 fixation for the efficiency of symbiotic nitrogen fixation in pea at vegetative growth and during pod formation.

PubMed

Fischinger, Stephanie Anastasia; Schulze, Joachim

2010-05-01

Nodule CO2 fixation is of pivotal importance for N2 fixation. The process provides malate for bacteroids and oxaloacetate for nitrogen assimilation. The hypothesis of the present paper was that grain legume nodules would adapt to higher plant N demand and more restricted carbon availability at pod formation through increased nodule CO2 fixation and a more efficient N2 fixation. Growth, N2 fixation, and nodule composition during vegetative growth and at pod formation were studied in pea plants (Pisum sativum L.). In parallel experiments, 15N2 and 13CO2 uptake, as well as nodule hydrogen and CO2 release, was measured. Plants at pod formation showed higher growth rates and N2 fixation per plant when compared with vegetative growth. The specific activity of active nodules was about 25% higher at pod formation. The higher nodule activity was accompanied by higher amino acid concentration in nodules and xylem sap with a higher share of asparagine. Nodule 13CO2 fixation was increased at pod formation, both per plant and per 15N2 fixed unit. However, malate concentration in nodules was only 40% of that during vegetative growth and succinate was no longer detectable. The data indicate that increased N2 fixation at pod formation is connected with strongly increased nodule CO2 fixation. While the sugar concentration in nodules at pod formation was not altered, the concentration of organic acids, namely malate and succinate, was significantly lower. It is concluded that strategies to improve the capability of nodules to fix CO2 and form organic acids might prolong intensive N2 fixation into the later stages of pod formation and pod filling in grain legumes.

Direct evidence that genetic variation in glycerol-3-phosphate and malate dehydrogenase genes (Gpdh and Mdh1) affects adult ethanol tolerance in Drosophila melanogaster.

PubMed

Eanes, Walter F; Merritt, Thomas J S; Flowers, Jonathan M; Kumagai, Seiji; Zhu, Chen-Tseh

2009-02-01

Many studies of alcohol adaptation in Drosophila melanogaster have focused on the Adh polymorphism, yet the metabolic elimination of alcohol should involve many enzymes and pathways. Here we evaluate the effects of glycerol-3-phosphate dehydrogenase (Gpdh) and cytosolic malate dehydrogenase (Mdh1) genotype activity on adult tolerance to ethanol. We have created a set of P-element-excision-derived Gpdh, Mdh1, and Adh alleles that generate a range of activity phenotypes from full to zero activity. Comparisons of paired Gpdh genotypes possessing 10 and 60% normal activity and 66 and 100% normal activity show significant effects where higher activity increases tolerance. Mdh1 null allele homozygotes show reductions in tolerance. We use piggyBac FLP-FRT site-specific recombination to create deletions and duplications of Gpdh. Duplications show an increase of 50% in activity and an increase of adult tolerance to ethanol exposure. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection related to adaptation to alcohols. Finally, we examine the interactions between activity genotypes for Gpdh, Mdh1, and Adh. We find no significant interlocus interactions. Observations on Mdh1 in both Gpdh and Adh backgrounds demonstrate significant increases in ethanol tolerance with partial reductions (50%) in cytosolic MDH activity. This observation strongly suggests the operation of pyruvate-malate and, in particular, pyruvate-citrate cycling in adaptation to alcohol exposure. We propose that an understanding of the evolution of tolerance to alcohols will require a system-level approach, rather than a focus on single enzymes.

Substrate specific effects of calcium on metabolism of rat heart mitochondria.

PubMed

Panov, A V; Scaduto, R C

1996-04-01

Oxidative metabolism in the heart is tightly coupled to mechanical work. Because this coupling process is believed to involve Ca2+, the roles of mitochondrial Ca2+ in the regulation of oxidative phosphorylation was studied in isolated rat heart mitochondria. The electrical component of the mitochondrial membrane potential (delta psi) and the redox state of the pyridine nucleotides were determined during the oxidation of various substrates under different metabolic states. In the absence of added adenine nucleotides, the NADP+ redox couple was almost completely reduced, regardless of the specific substrate and the presence of Ca2+, whereas NAD+ couple redox state was highly dependent on the substrate type and the presence of Ca2+. Titration of respiration with ADP, in the presence of excess hexokinase and glucose, showed that both respiration and NAD(P)+ reduction were very sensitive to ADP. The maximal enzyme reaction rate of ADP-stimulated respiration Michaelis constants (Km) for ADP were dependent on the particular substrate employed. delta psi was much less sensitive to ADP. With either alpha-ketoglutarate or glutamate as substrate, Ca2+ significantly increased reduction of NAD(P)+.Ca2+ did not influence NAD(P)+ reduction with either acetylcarnitine or pyruvate as substrate. In the presence of ADP, delta psi was increased by Ca2+ at all metabolic states with glutamate plus malate, 0.5 mM alpha-ketoglutarate plus malate, or pyruvate plus malate as substrates. The data presented support the hypothesis that cardiac respiration is controlled by the availability of both Ca2+ and ADP to mitochondria. The data indicate that an increase in substrate supply to mitochondria can increase mitochondrial respiration at given level of ADP. This effect can be produced by Ca2+ with substrates such as glutamate, which utilize alpha-ketoglutarate dehydrogenase activity for oxidation. Increases in respiration by Ca2+ may mitigate an increase in ADP during periods of increased cardiac work.

Stoichiometric Correlation of Malate Accumulation with Auxin-dependent K+-H+ Exchange and Growth in Avena Coleoptile Segments 12

PubMed Central

Haschke, Hans-Peter; Lüttge, Ulrich

1975-01-01

The action of auxin in the promotion of growth has been suggested in the literature to depend on cell wall acidification. In a former investigation by the present authors the electrochemical balance in auxin-induced proton extrusion was shown to be maintained by potassium net uptake. The present paper reports data demonstrating that the elongation of Avena coleoptile segments is accompanied by an accumulation of malate, which is stoichiometrically correlated with potassium uptake. We concluded that this malate accumulation is required in a mechanism regulating intracellular pH. PMID:16659374

Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast.

PubMed

Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H Ekkehard

2005-03-01

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH(-) to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.

Impaired pH Homeostasis in Arabidopsis Lacking the Vacuolar Dicarboxylate Transporter and Analysis of Carboxylic Acid Transport across the Tonoplast1

PubMed Central

Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H. Ekkehard

2005-01-01

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH− to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis. PMID:15728336

Organic Acids: The Pools of Fixed Carbon Involved in Redox Regulation and Energy Balance in Higher Plants

PubMed Central

Igamberdiev, Abir U.; Eprintsev, Alexander T.

2016-01-01

Organic acids are synthesized in plants as a result of the incomplete oxidation of photosynthetic products and represent the stored pools of fixed carbon accumulated due to different transient times of conversion of carbon compounds in metabolic pathways. When redox level in the cell increases, e.g., in conditions of active photosynthesis, the tricarboxylic acid (TCA) cycle in mitochondria is transformed to a partial cycle supplying citrate for the synthesis of 2-oxoglutarate and glutamate (citrate valve), while malate is accumulated and participates in the redox balance in different cell compartments (via malate valve). This results in malate and citrate frequently being the most accumulated acids in plants. However, the intensity of reactions linked to the conversion of these compounds can cause preferential accumulation of other organic acids, e.g., fumarate or isocitrate, in higher concentrations than malate and citrate. The secondary reactions, associated with the central metabolic pathways, in particularly with the TCA cycle, result in accumulation of other organic acids that are derived from the intermediates of the cycle. They form the additional pools of fixed carbon and stabilize the TCA cycle. Trans-aconitate is formed from citrate or cis-aconitate, accumulation of hydroxycitrate can be linked to metabolism of 2-oxoglutarate, while 4-hydroxy-2-oxoglutarate can be formed from pyruvate and glyoxylate. Glyoxylate, a product of either glycolate oxidase or isocitrate lyase, can be converted to oxalate. Malonate is accumulated at high concentrations in legume plants. Organic acids play a role in plants in providing redox equilibrium, supporting ionic gradients on membranes, and acidification of the extracellular medium. PMID:27471516

Combination of long noncoding RNA MALAT1 and carcinoembryonic antigen for the diagnosis of malignant pleural effusion caused by lung cancer.

PubMed

Wang, Wan-Wei; Zhou, Xi-Lei; Song, Ying-Jian; Yu, Chang-Hua; Zhu, Wei-Guo; Tong, Yu-Suo

2018-01-01

Long noncoding RNAs (lncRNAs) are present in body fluids, but their potential as tumor biomarkers has never been investigated in malignant pleural effusion (MPE) caused by lung cancer. The aim of this study was to assess the clinical significance of lncRNAs in pleural effusion, which could potentially serve as diagnostic and predictive markers for lung cancer-associated MPE (LC-MPE). RNAs from pleural effusion were extracted in 217 cases of LC-MPE and 132 cases of benign pleural effusion (BPE). Thirty-one lung cancer-associated lncRNAs were measured using quantitative real-time polymerase chain reaction (qRT-PCR). The level of carcinoembryonic antigen (CEA) was also determined. The receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were established to evaluate the sensitivity and specificity of the identified lncRNAs and other biomarkers. The correlations between baseline pleural effusion lncRNAs expression and response to chemotherapy were also analyzed. Three lncRNAs ( MALAT1 , H19 , and CUDR ) were found to have potential as diagnostic markers in LC-MPE. The AUCs for MALAT1 , H19 , CUDR , and CEA were 0.891, 0.783, 0.824, and 0.826, respectively. Using a logistic model, the combination of MALAT1 and CEA (AUC, 0.924) provided higher sensitivity and accuracy in predicting LC-MPE than CEA (AUC, 0.826) alone. Moreover, baseline MALAT1 expression in pleural fluid was inversely correlated with chemotherapy response in patients with LC-MPE. Pleural effusion lncRNAs were effective in differentiating LC-MPE from BPE. The combination of MALAT1 and CEA was more effective for LC-MPE diagnosis.

Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance.

PubMed

Liu, Jiping; Magalhaes, Jurandir V; Shaff, Jon; Kochian, Leon V

2009-02-01

Aluminum-activated root malate and citrate exudation play an important role in plant Al tolerance. This paper characterizes AtMATE, a homolog of the recently discovered sorghum and barley Al-tolerance genes, shown here to encode an Al-activated citrate transporter in Arabidopsis. Together with the previously characterized Al-activated malate transporter, AtALMT1, this discovery allowed us to examine the relationship in the same species between members of the two gene families for which Al-tolerance genes have been identified. AtMATE is expressed primarily in roots and is induced by Al. An AtMATE T-DNA knockdown line exhibited very low AtMATE expression and Al-activated root citrate exudation was abolished. The AtALMT1 AtMATE double mutant lacked both Al-activated root malate and citrate exudation and showed greater Al sensitivity than the AtALMT1 mutant. Therefore, although AtALMT1 is a major contributor to Arabidopsis Al tolerance, AtMATE also makes a significant but smaller contribution. The expression patterns of AtALMT1 and AtMATE and the profiles of Al-activated root citrate and malate exudation are not affected by the presence or absence of the other gene. These results suggest that AtALMT1-mediated malate exudation and AtMATE-mediated citrate exudation evolved independently to confer Al tolerance in Arabidopsis. However, a link between regulation of expression of the two transporters in response to Al was identified through work on STOP1, a transcription factor that was previously shown to be necessary for AtALMT1 expression. Here we show that STOP1 is also required for AtMATE expression and Al-activated citrate exudation.

NIP1;2 is a plasma membrane-localized transporter mediating aluminum uptake, translocation, and tolerance in Arabidopsis

PubMed Central

Wang, Yuqi; Li, Ruihong; Li, Demou; Jia, Xiaomin; Zhou, Dangwei; Li, Jianyong; Lyi, Sangbom M.; Hou, Siyu; Huang, Yulan

2017-01-01

Members of the aquaporin (AQP) family have been suggested to transport aluminum (Al) in plants; however, the Al form transported by AQPs and the roles of AQPs in Al tolerance remain elusive. Here we report that NIP1;2, a plasma membrane-localized member of the Arabidopsis nodulin 26-like intrinsic protein (NIP) subfamily of the AQP family, facilitates Al-malate transport from the root cell wall into the root symplasm, with subsequent Al xylem loading and root-to-shoot translocation, which are critical steps in an internal Al tolerance mechanism in Arabidopsis. We found that NIP1;2 transcripts are expressed mainly in the root tips, and that this expression is enhanced by Al but not by other metal stresses. Mutations in NIP1;2 lead to hyperaccumulation of toxic Al3+ in the root cell wall, inhibition of root-to-shoot Al translocation, and a significant reduction in Al tolerance. NIP1;2 facilitates the transport of Al-malate, but not Al3+ ions, in both yeast and Arabidopsis. We demonstrate that the formation of the Al-malate complex in the root tip apoplast is a prerequisite for NIP1;2-mediated Al removal from the root cell wall, and that this requires a functional root malate exudation system mediated by the Al-activated malate transporter, ALMT1. Taken together, these findings reveal a critical linkage between the previously identified Al exclusion mechanism based on root malate release and an internal Al tolerance mechanism identified here through the coordinated function of NIP1;2 and ALMT1, which is required for Al removal from the root cell wall, root-to-shoot Al translocation, and overall Al tolerance in Arabidopsis. PMID:28439024

Isotope effect studies of the chemical mechanism of nicotinamide adenine dinucleotide malic enzyme from Crassula

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grissom, C.B.; Willeford, O.; Wedding, R.T.

1987-05-05

The /sup 13/C primary kinetic isotope effect on the decarboxylation of malate by nicotinamide adenine dinucleotide malic enzyme from Crassula argentea is 1.0199 +/- 0.0006 with proteo L-malate-2-H and 1.0162 +/- 0.0003 with malate-2-d. The primary deuterium isotope effect is 1.45 +/- 0.10 on V/K and 1.93 +/- 0.13 on V/sub max/. This indicates a stepwise conversion of malate to pyruvate and CO/sub 2/ with hydride transfer preceding decarboxylation, thereby suggesting a discrete oxaloacetate intermediate. This is in agreement with the stepwise nature of the chemical mechanism of other malic enzymes despite the Crassula enzyme's inability to reduce or decarboxylatemore » oxaloacetate. Differences in morphology and allosteric regulation between enzymes suggest specialization of the Crassula malic enzyme for the physiology of crassulacean and acid metabolism while maintaining the catalytic events founds in malic enzymes from animal sources.« less

Interaction of E3 Ubiquitin Ligase MARCH7 with Long Noncoding RNA MALAT1 and Autophagy-Related Protein ATG7 Promotes Autophagy and Invasion in Ovarian Cancer.

PubMed

Hu, Jianguo; Zhang, Luo; Mei, Zhiqiang; Jiang, Yuan; Yi, Yuan; Liu, Li; Meng, Ying; Zhou, Lili; Zeng, Jianhua; Wu, Huan; Jiang, Xingwei

2018-05-22

Ubiquitin E3 ligase MARCH7 plays an important role in T cell proliferation and neuronal development. But its role in ovarian cancer remains unclear. This study aimed to investigate the role of Ubiquitin E3 ligase MARCH7 in ovarian cancer. Real-time PCR, immunohistochemistry and western blotting analysis were performed to determine the expression of MARCH7, MALAT1 and ATG7 in ovarian cancer cell lines and clinical specimens. The role of MARCH7 in maintaining ovarian cancer malignant phenotype was examined by Wound healing assay, Matrigel invasion assays and Mouse orthotopic xenograft model. Luciferase reporter assay, western blot analysis and ChIP assay were used to determine whether MARCH7 activates TGF-β-smad2/3 pathway by interacting with TGFβR2. MARCH7 interacted with MALAT1 by miR-200a (microRNA-200a). MARCH7 may function as a competing endogenous RNA (ceRNA) to regulate the expression of ATG7 by competing with miR-200a. MARCH7 regulated TGF-β-smad2/3 pathway by interacting with TGFβR2. Inhibition of TGF-β-smad2/3 pathway downregulated MARCH7, MALAT1 and ATG7. MiR-200a regulated TGF-β induced autophagy, invasion and metastasis of SKOV3 cells by targeting MARCH7. MARCH7 silencing inhibited autophagy invasion and metastasis of SKOV3 cells both in vitro and in vivo. In contrast, MARCH7 overexpression promoted TGF-β induced autophagy, invasion and metastasis of A2780 cells in vitro by depending on MALAT1 and ATG7. We also found that TGF-β-smad2/3 pathway regulated MARCH7 and ATG7 through MALAT1. These findings suggested that TGFβR2-Smad2/3-MALAT1/MARCH7/ATG7 feedback loop mediated autophagy, migration and invasion in ovarian cancer. © 2018 The Author(s). Published by S. Karger AG, Basel.

Two Members of the Aluminum-Activated Malate Transporter Family, SlALMT4 and SlALMT5, are Expressed during Fruit Development, and the Overexpression of SlALMT5 Alters Organic Acid Contents in Seeds in Tomato (Solanum lycopersicum).

PubMed

Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Nakano, Ryohei; Ushijima, Koichiro; Kubo, Yasutaka; Mori, Izumi C; Higashiizumi, Emi; Galis, Ivan; Yamamoto, Yoko

2016-11-01

The aluminum-activated malate transporter (ALMT) family of proteins transports malate and/or inorganic anions across plant membranes. To demonstrate the possible role of ALMT genes in tomato fruit development, we focused on SlALMT4 and SlALMT5, the two major genes expressed during fruit development. Predicted proteins were classified into clade 2 of the family, many members of which localize to endomembranes. Tissue-specific gene expression was determined using transgenic tomato expressing the β-glucuronidase reporter gene controlled by their own promoters. Both the genes were expressed in vascular bundles connecting to developing seeds in fruit and in the embryo of mature seeds. Further, SlALMT5 was expressed in embryo in developing seeds in fruit. Subcellular localization of both proteins to the endoplasmic reticulum (ER) was established by transiently expressing the green fluorescent protein fusions in plant protoplasts. SlALMT5 probably localized to other endomembranes as well. Localization of SlALMT5 to the ER was also confirmed by immunoblot analysis. The transport function of both SlALMT proteins was investigated electrophysiologically in Xenopus oocytes. SlALMT5 transported malate and inorganic anions such as nitrate and chloride, but not citrate. SlALMT4 also transported malate, but the results were less consistent perhaps because it did not localize strongly to the plasma membrane. To elucidate the physiological role of SlALMT5 further, we overexpressed SlALMT5 in tomato. Compared with the wild type, overexpressors exhibited higher malate and citrate contents in mature seeds, but not in fruit. We conclude that the malate transport function of SlALMT5 expressed in developing fruit influences the organic acid contents in mature seeds. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Systemic Sunitinib Malate Treatment for Advanced Juxtapapillary Retinal Hemangioblastomas Associated with von Hippel-Lindau Disease

PubMed Central

Knickelbein, Jared E.; Jacobs-El, Naima; Wong, Wai T.; Wiley, Henry E.; Cukras, Catherine A.; Meyerle, Catherine B.; Chew, Emily Y.

2016-01-01

Purpose To describe the clinical course of advanced juxtapapillary retinal capillary hemangioblastomas (RCH) associated with von Hippel-Lindau (VHL) disease treated with systemic sunitinib malate, an agent that inhibits both anti-vascular endothelial growth factor and anti-platelet-derived growth factor signaling. Design Observational case review. Participants Three patients with advanced VHL-related juxtapapillary RCH treated with systemic sunitinib malate. Methods Patient 1 was followed routinely every 4 months while on systemic sunitinib prescribed by her oncologist for metastatic pancreatic neuroendocrine and kidney tumors. Patients 2 and 3 were part of a prospective clinical trial evaluating the use of systemic sunitinib for ocular VHL lesions during a period of 9 months. Visual acuity, size of RCH, and degree of exudation were recorded at each visit. Optical coherence tomography (OCT) and fluorescein angiography were also obtained at some visits. Main Outcome Measures Visual acuity, size of RCH, and degree of exudation. Results Three patients with advanced VHL-associated juxtapapillary RCH were treated with systemic sunitinib malate. While none of the patients lost vision during therapy, treatment with sunitinib malate did not improve visual acuity or reduce the size of RCH. Improvements in RCH-associated retinal edema were observed in two patients. All patients experienced multiple adverse effects, including thyroid toxicity, thrombocytopenia, nausea, fatigue, jaundice, and muscle aches. Two of the three patients had to discontinue treatment prematurely and the third required dose reduction. Conclusions Systemic sunitinib malate may be useful in slowing progression of ocular disease from VHL-associated RCH. However, significant systemic adverse effects limited its use in this small series, and systemic sunitinib malate may not be safe for treatment of RCH when used at the doses described in this report. Further studies are required to determine if this medication used at lower doses with different treatment strategies, other medications in the same class or drugs directed at multiple targets in the tumor, may be safer and more effective for the treatment of advanced VHL-associated RCH. PMID:28670632

Polyesters from microorganisms.

PubMed

Kim, Y B; Lenz, R W

2001-01-01

Bacterial polyesters have been found to have useful properties for applications as thermoplastics, elastomers, and adhesives and are biodegradable and biocompatible. Poly(3-hydroxyalkanoates) (PHAs) and poly(beta-malate) are the most representative polyesters synthesized by microorganisms. PHAs containing a wide variety of repeating units can be produced by bacteria, including those containing many types of pendant functional groups which can be synthesized by microorganisms that are grown on unnatural organic substrates. Poly(beta-malate) is of interest primarily for medical applications, especially for drug delivery systems. In this chapter, the bacterial production and properties of poly(3-hydroxyalkanoates) and poly(beta-malate) are described with emphasis on the former.

Dietary keto-acid feed-back on pituitary activity in gilthead sea bream: effects of oral doses of AKG. A proteomic approach.

PubMed

Ibarz, Antoni; Costa, Rita; Harrison, Adrian P; Power, Deborah M

2010-12-01

The influence of a daily oral dose of alpha-ketoglutarate (AKG, 0.1 g/kg body weight), an intermediate metabolite in the Krebs cycle and a dietary additive, on the pituitary proteome of gilthead sea bream was determined by two-dimensional electrophoresis (2-DE). A high-resolution map of the sea bream pituitary proteome was generated. Proteins with a modified expression between Controls and AKG treated fish were further analysed by MALDI-TOF/TOF-MS and liquid chromatography combined with a nanoelectrospray (LC-MS/MS). The main changes in the proteome induced by AKG treatment were grouped. Metabolic proteins up-regulated with AKG supplementation included fructose-bis-phosphate aldolase, glyceraldehyde-phosphate dehydrogenase and malate dehydrogenase, all related to glucose metabolism (p<0.000). Protein folding related up-regulation with AKG supplementation included two isoforms of heat shock proteins as well as cyclophylin and chaperonin (p<0.000). An unexpected form of apolipoprotein-A-1 with lower molecular weight (15-16 kDa) was evidenced as being highly abundant in the pituitary proteome of Controls, yet it was down-regulated by AKG treatment. Finally, proteins found to be associated with regeneration of neural function namely cofilin and Vat-protein were up-regulated after AKG supplementation. The only hormone to be modified by AKG treatment was somatolactin, which was significantly down-regulated cf. Controls. In summary, these results provide evidence of a potential endocrine/metabolic regulatory loop activated by AKG supplementation. Copyright © 2010 Elsevier Inc. All rights reserved.

Occurrence of the malate-aspartate shuttle in various tumor types.

PubMed

Greenhouse, W V; Lehninger, A L

1976-04-01

The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm.

Inhibition of several enzymes by gold compounds. II. beta-Glucuronidase, acid phosphatase and L-malate dehydrogenase by sodium thiomalatoraurate (I), sodium thiosulfatoaurate (I) and thioglucosoaurate (I).

PubMed

Lee, M T; Ahmed, T; Haddad, R; Friedman, M E

1989-01-01

Bovine liver beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32), wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) and bovine liver L-malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) were inhibited by a series of gold (I) complexes that have been used as anti-inflammatory drugs. Both sodium thiosulfatoaurate (I) (Na AuTs) and sodium thiomalatoraurate (NaAuTM) effectively inhibited all three enzymes, while thioglucosoaurate (I) (AuTG) only inhibited L-malate dehydrogenase. The equilibrium constants (K1) ranged from nearly 4000 microM for the NaAuTM-beta-glucuronidase interaction to 24 microM for the NaAuTS-beta-glucuronidase interaction. The rate of covalent bond formation (kp) ranged from 0.00032 min-1 for NaAuTM-beta-glucuronidase formation to 1.7 min-1 for AuTG-L-malate dehydrogenase formation. The equilibrium data shows that the gold (I) drugs bind by several orders lower than the gold (III) compounds, suggesting a significantly stronger interaction between the more highly charged gold ion and the enzyme. Yet the rate of covalent bond formation depends as much on the structure of the active site as upon the lability of the gold-ligand bond. It was also observed that the more effective the gold inhibition the more toxic the compound.

Expression of codon-optmized phosphoenolpyruvate carboxylase gene from Glaciecola sp. HTCC2999 in Escherichia coli and its application for C4 chemical production.

PubMed

Park, Soohyun; Pack, Seung Pil; Lee, Jinwon

2012-08-01

We examined the expression of the phosphoenolpyruvate carboxylase (PEPC) gene from marine bacteria in Escherichia coli using codon optimization. The codon-optimized PEPC gene was expressed in the E. coli K-12 strain W3110. SDS-PAGE analysis revealed that the codon-optimized PEPC gene was only expressed in E. coli, and measurement of enzyme activity indicated the highest PEPC activity in the E. coli SGJS112 strain that contained the codon-optimized PEPC gene. In fermentation assays, the E. coli SGJS112 produced the highest yield of oxaloacetate using glucose as the source and produced a 20-times increase in the yield of malate compared to the control. We concluded that the codon optimization enabled E. coli to express the PEPC gene derived from the Glaciecola sp. HTCC2999. Also, the expressed protein exhibited an enzymatic activity similar to that of E. coli PEPC and increased the yield of oxaloacetate and malate in an E. coli system.

Proteomic analysis of a high aluminum tolerant yeast Rhodotorula taiwanensis RS1 in response to aluminum stress.

PubMed

Wang, Chao; Wang, Chang Yi; Zhao, Xue Qiang; Chen, Rong Fu; Lan, Ping; Shen, Ren Fang

2013-10-01

Rhodotorula taiwanensis RS1 is a high-aluminum (Al)-tolerant yeast that can survive in Al concentrations up to 200mM. The mechanisms for the high Al tolerance of R. taiwanensis RS1 are not well understood. To investigate the molecular mechanisms underlying Al tolerance and toxicity in R. taiwanensis RS1, Al toxicity-induced changes in the total soluble protein profile were analyzed using two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry. A total of 33 differentially expressed proteins responding to Al stress were identified from approximately 850 reproducibly detected proteins. Among them, the abundance of 29 proteins decreased and 4 increased. In the presence of 100mM Al, the abundance of proteins involved in DNA transcription, protein translation, DNA defense, Golgi functions and glucose metabolism was decreased. By contrast, Al treatment led to increased abundance of malate dehydrogenase, which correlated with increased malate dehydrogenase activity and the accumulation of intracellular citrate, suggesting that Al-induced intracellular citrate could play an important role in detoxification of Al in R. taiwanensis RS1. © 2013.

Caloric restriction counteracts age-related changes in the activities of sorbitol metabolizing enzymes from mouse liver

PubMed Central

Hagopian, Kevork; Ramsey, Jon J.; Weindruch, Richard

2009-01-01

The influence of caloric restriction (CR) on hepatic sorbitol-metabolizing enzyme activities was investigated in young and old mice. Aldose reductase and sorbitol dehydrogenase activities were significantly lower in old CR mice than in old controls. Young CR mice showed decreased aldose reductase activity and a trend towards decreased sorbitol dehydrogenase when compared to controls. Metabolites of the pathway, namely sorbitol, glucose and fructose were decreased by CR in young and old mice. Pyruvate levels were decreased by CR in both young and old mice, while lactate decreased only in old CR. Malate levels increased in old CR but remained unchanged in young CR, when compared with controls. Accordingly, the lactae/pyruvate and malate/pyruvate ratios in young and old CR mice were increased, indicating increased NADH/NAD and NADPH/NADP redox couples, respectively. The results indicate that decreased glucose levels under CR conditions lead to decreased sorbitol pathway enzyme activities and metabolite levels, and could contribute to the beneficial effects of long-term CR through decreased sorbitol levels and NADPH sparing. PMID:18953666

Role of malate dehydrogenase in facilitating lactate dehydrogenase to support the glycolysis pathway in tumors.

PubMed

Mansouri, Siavash; Shahriari, Ali; Kalantar, Hadi; Moini Zanjani, Taraneh; Haghi Karamallah, Mojtaba

2017-04-01

High aerobic glycolysis, as one of the hallmarks of cancer cells, requires nicotinamide adenine dinucleotide (NAD + ) as a vital co-factor, to guarantee the flow of glycolysis. Malate dehydrogenase (MDH), as an important enzyme in cancer metabolism, is a source of NAD + additional to lactate dehydrogenase (LDH). The current study aimed to elucidate the kinetic parameters of MDH in human breast cancer and evaluate its supportive role in the glycolysis pathway. The Michaelis-Menten constant (K m ) and maximum velocity (V max ) of MDH were determined in the crude extracts of human breast tumors and healthy tissue samples, which were obtained directly from the operating theatre. To assess the potential role of MDH in supporting glycolysis, the MDH activity was measured when the LDH activity was inhibited by different concentrations of oxamate, an inhibitor of LDH in breast cancer cell lines. The K m of cancerous MDH (C-MDH) was the same as the healthy MDH, although the V max of C-MDH was higher relative to the healthy MDH. Notably, the MDH activity was increased in the MDA-MB-231 cell line, which was treated with the LDH inhibitor (oxamate), but not in the MCF-7 cell line (P<0.05). The higher tendency of C-MDH for NAD + and malate generation in cancer cells is an effective approach for supporting glycolysis. Increasing MDH activity in the absence of LDH demonstrates the supportive role of MDH in glycolysis. Therefore, decreasing MDH activity and expression in a forward reaction may present as a valid molecular target to abolish its potential effect on tumor metabolism.

Developmental Physiology of Cluster-Root Carboxylate Synthesis and Exudation in Harsh Hakea. Expression of Phosphoenolpyruvate Carboxylase and the Alternative Oxidase1

PubMed Central

Shane, Michael W.; Cramer, Michael D.; Funayama-Noguchi, Sachiko; Cawthray, Gregory R.; Millar, A. Harvey; Day, David A.; Lambers, Hans

2004-01-01

Harsh hakea (Hakea prostrata R.Br.) is a member of the Proteaceae family, which is highly represented on the extremely nutrient-impoverished soils in southwest Australia. When phosphorus is limiting, harsh hakea develops proteoid or cluster roots that release carboxylates that mobilize sparingly soluble phosphate in the rhizosphere. To investigate the physiology underlying the synthesis and exudation of carboxylates from cluster roots in Proteaceae, we measured O2 consumption, CO2 release, internal carboxylate concentrations and carboxylate exudation, and the abundance of the enzymes phosphoenolpyruvate carboxylase and alternative oxidase (AOX) over a 3-week time course of cluster-root development. Peak rates of citrate and malate exudation were observed from 12- to 13-d-old cluster roots, preceded by a reduction in cluster-root total protein levels and a reduced rate of O2 consumption. In harsh hakea, phosphoenolpyruvate carboxylase expression was relatively constant in cluster roots, regardless of developmental stage. During cluster-root maturation, however, the expression of AOX protein increased prior to the time when citrate and malate exudation peaked. This increase in AOX protein levels is presumably needed to allow a greater flow of electrons through the mitochondrial electron transport chain in the absence of rapid ATP turnover. Citrate and isocitrate synthesis and accumulation contributed in a major way to the subsequent burst of citrate and malate exudation. Phosphorus accumulated by harsh hakea cluster roots was remobilized during senescence as part of their efficient P cycling strategy for growth on nutrient impoverished soils. PMID:15122030

Phosphoenolpyruvate carboxykinase and gluconeogenesis in grape pericarp.

PubMed

Walker, Robert P; Battistelli, Alberto; Moscatello, Stefano; Técsi, László; Leegood, Richard C; Famiani, Franco

2015-12-01

Glycolysis from sugars is necessary at all stages of development of grape pericarp, and this raises the question as to why gluconeogenesis from malate occurs. Phosphoenolpyruvate carboxykinase (PEPCK) is required for gluconeogenesis in grape pericarp. In this study we determined the abundance of PEPCK protein and activity in different parts of grape pericarp during its development. Both PEPCK protein and activity were present throughout development, however, in both the skin and the flesh their abundance increased greatly at the start of ripening. This coincided with the onset of the decrease in the malate content of the berry. The location of PEPCK in the pericarp at different stages of development was determined using both immunohistochemistry and dissection. We provide a possible explanation for the occurrence of gluconeogenesis in grape pericarp. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

A Bacillus subtilis malate dehydrogenase gene.

PubMed Central

Jin, S; De Jesús-Berríos, M; Sonenshein, A L

1996-01-01

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation. PMID:8550482

Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

USDA-ARS?s Scientific Manuscript database

Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weissmann, Sarit; Ma, Fangfang; Furuyama, Koki

C 4 photosynthesis in grasses requires the coordinated movement of metabolites through two specialized leaf cell types, mesophyll (M) and bundle sheath (BS), to concentrate CO 2 around Rubisco. Despite the importance of transporters in this process, few have been identified or rigorously characterized. In maize (Zea mays), DCT2 has been proposed to function as a plastid-localizedmalate transporter and is preferentially expressed in BS cells. Here, we characterized the role of DCT2 in maize leaves using Activator-tagged mutant alleles. Our results indicate that DCT2 enables the transport of malate into the BS chloroplast. Isotopic labeling experiments show that the lossmore » of DCT2 results in markedly different metabolic network operation and dramatically reduced biomass production. In the absence of a functioning malate shuttle, dct2 lines survive through the enhanced use of the phosphoenolpyruvate carboxykinase carbon shuttle pathway that in wild-type maize accounts for ;25% of the photosynthetic activity. The results emphasize the importance of malate transport during C 4 photosynthesis, define the role of a primary malate transporter in BS cells, and support a model for carbon exchange between BS and M cells in maize.« less

Assessment the levels of tartrate-resistant acid phosphatase (TRAP) on mice fed with eggshell calcium citrate malate.

PubMed

Yu, Yiding; Zhang, Mingdi; Lin, Songyi; Wang, Liyan; Liu, Jingbo; Jones, Gregory; Huang, Hsiang-Chi

2013-07-01

Optimized conditions were obtained by one-factor-at-a-time test (OFAT) and ternary quadratic regression orthogonal composite design (TQROCD) respectively. By pulse electric fields (PEF) technology, the process of eggshell calcium citrate malate (ESCCM), eggshell calcium citrate (ESCC) and eggshells calcium malate (ESCM) were comprehensive compared. The levels of tartrate-resistant acid phosphatase (TRAP) and the bioavailability on mice fed with eggshell calcium citrate malate (ESCCM) treated by pulsed electric field (PEF) were evaluated. Results showed that the rates of calcium dissolution of the different acids studied can be arranged as ESCCM (7.90 mg/mL)>ESCC (7.12 mg/mL)>ESCM (7.08 mg/mL) from highest to lowest rate of dissolution. At the same dose 133.0 mg kg(-1) d(-1), the levels of TRAP in the ESCCM treatment groups were significantly lower than those in ESCM and ESCC (P<0.05). Bone calcium content in the mice fed with ESCCM was generally higher than fed with ESCM and ESCC. Copyright © 2013 Elsevier B.V. All rights reserved.

Mitochondrial redox cycling of mitoquinone leads to superoxide production and cellular apoptosis.

PubMed

Doughan, Abdulrahman K; Dikalov, Sergey I

2007-11-01

The mitochondria-targeted drug mitoquinone (MitoQ) has been used as an antioxidant that may selectively block mitochondrial oxidative damage; however, it has been recently suggested to increase reactive oxygen species (ROS) generation in malate- and glutamate-fueled mitochondria. To address this controversy, we studied the effects of MitoQ on endothelial and mitochondrial ROS production. We found that in a cell-free system with flavin-containing enzyme cytochrome P-450 reductase, MitoQ is a very efficient redox cycling agent and produced more superoxide compared with equal concentrations of menadione (10-1,000 nM). Treatment of endothelial cells with MitoQ resulted in a dramatic increase in superoxide production. In isolated mitochondria, MitoQ increased complex I-driven mitochondrial ROS production, whereas supplementation with ubiquinone-10 had no effect on ROS production. Similar results were observed in mitochondria isolated from endothelial cells incubated for 1 h with MitoQ. Inhibitor analysis suggested that the redox cycling of MitoQ occurred at two sites on complex I, proximal and distal to the rotenone-binding site. This was confirmed by demonstrating the redox cycling of MitoQ on purified mitochondrial complex I as well as NADH-fueled submitochondrial particles. Mitoquinone time- and dose-dependently increased endothelial cell apoptosis. These findings demonstrate that MitoQ may be prooxidant and proapoptotic because its quinone group can participate in redox cycling and superoxide production. In light of these results, studies using mitoquinone as an antioxidant should be interpreted with caution.

Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

PubMed

Ramesh, Sunita A; Kamran, Muhammad; Sullivan, Wendy; Chirkova, Larissa; Okamoto, Mamoru; Degryse, Fien; McLaughlin, Michael; Gilliham, Matthew; Tyerman, Stephen D

2018-05-01

Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABA A receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA] i ) in both wheat ( Triticum aestivum ) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA] i because TaALMT1 facilitates GABA efflux but GABA does not complex Al 3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14 C-GABA uptake into TaALMT1 -expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1 F213C ) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA] i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status. © 2018 American Society of Plant Biologists. All rights reserved.

Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II, and the identity of the ligand in the "oxaloacetate-inhibited" state.

PubMed

Huang, Li-Shar; Shen, John T; Wang, Andy C; Berry, Edward A

2006-01-01

Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115-1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the "malate-like intermediate" found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108-1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y.O. Belikova, et al., Biochim. Biophys. Acta 936 1-9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.

Crystallographic Studies of the Binding of Ligands to theDicarboxylate Site of Complex II, and the Identity of the Ligand in the'Oxaloacetate-Inhibited' State

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Li-Shar; Shen, John T.; Wang, Andy C.

2006-07-01

Mitochondrial Complex II (succinate:ubiquinoneoxidoreductase) is purified in a partially innactivated state, which canbe activated by removal of tightly bound oxaloacetate (Kearney, E.B. etal. Biochem Biophys Res Commun 49, 1115-1121). We crystallized Complex IIin the presence of oxaloacetate or with the endogenous inhibitor bound.The structure showed a ligand essentially identical to the "malate-likeintermediate" found in Shewanella Flavocytochrome c crystallized withfumarate (Taylor, P., et al. Nat Struct Biol 6, 1108-1112.)Crystallization of Complex II in the presence of excess fumarate alsogave the malate-like intermediate or a mixture of that and fumarate atthe active site. In order to more conveniently monitor the occupationstate ofmore » the dicarboxylate site, we are developing a library of UV/Visspectral effects induced by binding different ligands to the site.Treatment with fumarate results in rapid development of the fumaratedifference spectrum and then a very slow conversion into a speciesspectrally similar to the OAA liganded complex. Complex II is known to becapable of oxidizing malate to the enol form of oxaloacetate (Belikova,Y.O., et al. Biochim Biophys Acta 936, 1-9). The observations abovesuggest it may also be capable of interconverting fumarate and malate. Itmay be useful for understanding the mechanism and regulation of theenzyme to identify the malate-like intermediate and its pathway offormation from oxaloacetate or fumarate.« less

A co-expression gene network associated with developmental regulation of apple fruit acidity.

PubMed

Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Xu, Kenong

2015-08-01

Apple fruit acidity, which affects the fruit's overall taste and flavor to a large extent, is primarily determined by the concentration of malic acid. Previous studies demonstrated that the major QTL malic acid (Ma) on chromosome 16 is largely responsible for fruit acidity variations in apple. Recent advances suggested that a natural mutation that gives rise to a premature stop codon in one of the two aluminum-activated malate transporter (ALMT)-like genes (called Ma1) is the genetic causal element underlying Ma. However, the natural mutation does not explain the developmental changes of fruit malate levels in a given genotype. Using RNA-seq data from the fruit of 'Golden Delicious' taken at 14 developmental stages from 1 week after full-bloom (WAF01) to harvest (WAF20), we characterized their transcriptomes in groups of high (12.2 ± 1.6 mg/g fw, WAF03-WAF08), mid (7.4 ± 0.5 mg/g fw, WAF01-WAF02 and WAF10-WAF14) and low (5.4 ± 0.4 mg/g fw, WAF16-WAF20) malate concentrations. Detailed analyses showed that a set of 3,066 genes (including Ma1) were expressed not only differentially (P FDR < 0.05) between the high and low malate groups (or between the early and late developmental stages) but also in significant (P < 0.05) correlation with malate concentrations. The 3,066 genes fell in 648 MapMan (sub-) bins or functional classes, and 19 of them were significantly (P FDR < 0.05) co-enriched or co-suppressed in a malate dependent manner. Network inferring using the 363 genes encompassed in the 19 (sub-) bins, identified a major co-expression network of 239 genes. Since the 239 genes were also differentially expressed between the early (WAF03-WAF08) and late (WAF16-WAF20) developmental stages, the major network was considered to be associated with developmental regulation of apple fruit acidity in 'Golden Delicious'.

The crystal structures of the tri-functional Chloroflexus aurantiacus and bi-functional Rhodobacter sphaeroides malyl-CoA lyases and comparison with CitE-like superfamily enzymes and malate synthases.

PubMed

Zarzycki, Jan; Kerfeld, Cheryl A

2013-11-09

Malyl-CoA lyase (MCL) is a promiscuous carbon-carbon bond lyase that catalyzes the reversible cleavage of structurally related Coenzyme A (CoA) thioesters. This enzyme plays a crucial, multifunctional role in the 3-hydroxypropionate bi-cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus. A second, phylogenetically distinct MCL from Rhodobacter sphaeroides is involved in the ethylmalonyl-CoA pathway for acetate assimilation. Both MCLs belong to the large superfamily of CitE-like enzymes, which includes the name-giving β-subunit of citrate lyase (CitE), malyl-CoA thioesterases and other enzymes of unknown physiological function. The CitE-like enzyme superfamily also bears sequence and structural resemblance to the malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or both (the malate synthases). Here we report the first crystal structures of MCLs from two different phylogenetic subgroups in apo- and substrate-bound forms. Both the C. aurantiacus and the R. sphaeroides MCL contain elaborations on the canonical β8/α8 TIM barrel fold and form hexameric assemblies. Upon ligand binding, changes in the C-terminal domains of the MCLs result in closing of the active site, with the C-terminal domain of one monomer forming a lid over and contributing side chains to the active site of the adjacent monomer. The distinctive features of the two MCL subgroups were compared to known structures of other CitE-like superfamily enzymes and to malate synthases, providing insight into the structural subtleties that underlie the functional versatility of these enzymes. Although the C. aurantiacus and the R. sphaeroides MCLs have divergent primary structures (~37% identical), their tertiary and quaternary structures are very similar. It can be assumed that the C-C bond formation catalyzed by the MCLs occurs as proposed for malate synthases. However, a comparison of the two MCL structures with known malate synthases raised the question why the MCLs are not also able to hydrolyze CoA thioester bonds. Our results suggest the previously proposed reaction mechanism for malate synthases may be incomplete or not entirely correct. Further studies involving site-directed mutagenesis based on these structures may be required to solve this puzzling question.

Peripheral Effects of FAAH Deficiency on Fuel and Energy Homeostasis: Role of Dysregulated Lysine Acetylation

PubMed Central

Vaitheesvaran, Bhavapriya; Yang, Li; Hartil, Kirsten; Glaser, Sherrye; Yazulla, Stephen; Bruce, James E.; Kurland, Irwin J.

2012-01-01

Background FAAH (fatty acid amide hydrolase), primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA). Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH−/− mice. Methodology/Principal Findings FAAH−/− mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry). FAAH−/− mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN). Fed state skeletal muscle and liver triglyceride levels was increased 2–3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH−/− mice. Dysregulated hepatic FAAH−/− lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH−/− acetyl-CoA (85%, p<0.01) corresponded to similar increases in citrate levels (45%). Altered FAAH−/− mitochondrial malate dehydrogenase (MDH2) acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH−/− mice was consistent with a compensating contribution from decreased acetylation of fed FAAH−/− aldolase B. Fed FAAH−/− alcohol dehydrogenase (ADH) acetylation was also decreased. Conclusions/Significance Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH−/− mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver's role in fostering the pre-diabetic state, and may reflect part of the mechanism underlying the hepatic effects of endocannabinoids in alcoholic liver disease mouse models. PMID:22442717

Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheat

USDA-ARS?s Scientific Manuscript database

In this study we examined the role of protein phosphorylation & dephosphorylation in the transport properties of the wheat root malate efflux transporter underlying Al resistance, TaALMT1. Preincubation of Xenopus laevis oocytes expressing TaALMT1 with protein kinase inhibitors (K252a and staurospo...

Does Citrulline Malate Enhance Physical Performance

DTIC Science & Technology

2010-10-01

Jakeman, P.M. (2010). Citrulline malate enhances athletic anaerobic performance and relieves muscle soreness. Journal of Strength and Conditioning...returned for their third and final VO2 max test. VO2max, lactate threshold , maximum watts reached, ratings of perceived exertion and pre- and post...7 Figure 7. Lactate threshold (as % of VO2max) for all three conditions

Regulation of Stomatal Immunity by Interdependent Functions of a Pathogen-Responsive MPK3/MPK6 Cascade and Abscisic Acid

PubMed Central

Zhang, Lawrence; Sun, Tiefeng

2017-01-01

Activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses after plants sense an invading pathogen. Here, we show that MPK3 and MPK6, two Arabidopsis thaliana pathogen-responsive MAPKs, and their upstream MAPK kinases, MKK4 and MKK5, are essential to both stomatal and apoplastic immunity. Loss of function of MPK3 and MPK6, or their upstream MKK4 and MKK5, abolishes pathogen/microbe-associated molecular pattern- and pathogen-induced stomatal closure. Gain-of-function activation of MPK3/MPK6 induces stomatal closure independently of abscisic acid (ABA) biosynthesis and signaling. In contrast, exogenously applied organic acids such as malate or citrate are able to reverse the stomatal closure induced by MPK3/MPK6 activation. Gene expression analysis and in situ enzyme activity staining revealed that malate metabolism increases in guard cells after activation of MPK3/MPK6 or inoculation of pathogen. In addition, pathogen-induced malate metabolism requires functional MKK4/MKK5 and MPK3/MPK6. We propose that the pathogen-responsive MPK3/MPK6 cascade and ABA are two essential signaling pathways that control, respectively, the organic acid metabolism and ion channels, two main branches of osmotic regulation in guard cells that function interdependently to control stomatal opening/closure. PMID:28254778

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves. Kinetic properties of different oligomeric structures.

PubMed

Iglesias, A A; Andreo, C S

1990-09-24

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves was inhibited by increasing the ionic strength in the assay medium. The inhibitory effect was higher at pH 7.0 than 8.0, with median inhibitory concentrations (IC50) of 89 mM and 160 mM respectively, for inhibition by NaCl. Gel-filtration experiments indicated that the enzyme dissociated into dimers and monomers when exposed to high ionic strength (0.3 M NaCl). By using the enzyme-dilution approach in the absence and presence of 0.3 M NaCl, the kinetic properties of each oligomeric species of the protein was determined at pH 7.0 and 8.0. Tetrameric, dimeric and monomeric structures were shown to be active but with different V and Km values. The catalytic efficiency of the oligomers was tetramer greater than dimer greater than monomer, and each quaternary structure exhibited higher activity at pH 8.0 than 7.0. Dissociation constants for the equilibria between the different oligomeric forms of the enzyme were determined. It was established that Kd values were affected by pH and Mg2+ levels in the medium. Results suggest that the distinct catalytic properties of the different oligomeric forms of NADP-dependent malate dehydrogenase and changes in their equilibrium could be the molecular basis for an efficient physiological regulation of the decarboxylation step of C4 metabolism.

Carbon monoxide improves neuronal differentiation and yield by increasing the functioning and number of mitochondria.

PubMed

Almeida, Ana S; Sonnewald, Ursula; Alves, Paula M; Vieira, Helena L A

2016-08-01

The process of cell differentiation goes hand-in-hand with metabolic adaptations, which are needed to provide energy and new metabolites. Carbon monoxide (CO) is an endogenous cytoprotective molecule able to inhibit cell death and improve mitochondrial metabolism. Neuronal differentiation processes were studied using the NT2 cell line, which is derived from human testicular embryonic teratocarcinoma and differentiates into post-mitotic neurons upon retinoic acid treatment. CO-releasing molecule A1 (CORM-A1) was used do deliver CO into cell culture. CO treatment improved NT2 neuronal differentiation and yield, since there were more neurons and the total cell number increased following the differentiation process. CO supplementation enhanced the mitochondrial population in post-mitotic neurons derived from NT2 cells, as indicated by an increase in mitochondrial DNA. CO treatment during neuronal differentiation increased the extent of the classical metabolic change that occurs during neuronal differentiation, from glycolytic to more oxidative metabolism, by decreasing the ratio of lactate production and glucose consumption. The expression of pyruvate and lactate dehydrogenases was higher, indicating an augmented oxidative metabolism. Moreover, these findings were corroborated by an increased percentage of (13) C incorporation from [U-(13) C]glucose into the tricarboxylic acid cycle metabolites malate and citrate, and also glutamate and aspartate in CO-treated cells. Finally, under low levels of oxygen (5%), which enhances glycolytic metabolism, some of the enhancing effects of CO on mitochondria were not observed. In conclusion, our data show that CO improves neuronal and mitochondrial yield by stimulation of tricarboxylic acid cycle activity, and thus oxidative metabolism of NT2 cells during the process of neuronal differentiation. The process of cell differentiation is coupled with metabolic adaptations. Carbon monoxide (CO) is an endogenous cytoprotective gasotransmitter able to prevent cell death and improve mitochondrial metabolism. Herein CO supplementation improved neuronal differentiation yield, by enhancing mitochondrial population and promoting the classical metabolic change that occurs during neuronal differentiation, from glycolytic to oxidative metabolism. © 2016 International Society for Neurochemistry.

Kinetic model of mitochondrial Krebs cycle: unraveling the mechanism of salicylate hepatotoxic effects.

PubMed

Mogilevskaya, Ekaterina; Demin, Oleg; Goryanin, Igor

2006-10-01

This paper studies the effect of salicylate on the energy metabolism of mitochondria using in silico simulations. A kinetic model of the mitochondrial Krebs cycle is constructed using information on the individual enzymes. Model parameters for the rate equations are estimated using in vitro experimental data from the literature. Enzyme concentrations are determined from data on respiration in mitochondrial suspensions containing glutamate and malate. It is shown that inhibition in succinate dehydrogenase and alpha-ketoglutarate dehydrogenase by salicylate contributes substantially to the cumulative inhibition of the Krebs cycle by salicylates. Uncoupling of oxidative phosphorylation has little effect and coenzyme A consumption in salicylates transformation processes has an insignificant effect on the rate of substrate oxidation in the Krebs cycle. It is found that the salicylate-inhibited Krebs cycle flux can be increased by flux redirection through addition of external glutamate and malate, and depletion in external alpha-ketoglutarate and glycine concentrations.

Salmonella enterica serovar Typhimurium mutants unable to convert malate to pyruvate and oxaloacetate are avirulent and immunogenic in BALB/c mice.

PubMed

Mercado-Lubo, Regino; Leatham, Mary P; Conway, Tyrrell; Cohen, Paul S

2009-04-01

Previously, we showed that the Salmonella enterica serovar Typhimurium SR-11 tricarboxylic acid (TCA) cycle must operate as a complete cycle for full virulence after oral infection of BALB/c mice (M. Tchawa Yimga, M. P. Leatham, J. H. Allen, D. C. Laux, T. Conway, and P. S. Cohen, Infect. Immun. 74:1130-1140, 2006). In the same study, we showed that for full virulence, malate must be converted to both oxaloacetate and pyruvate. Moreover, it was recently demonstrated that blocking conversion of succinyl-coenzyme A to succinate attenuates serovar Typhimurium SR-11 but does not make it avirulent; however, blocking conversion of succinate to fumarate renders it completely avirulent and protective against subsequent oral infection with the virulent serovar Typhimurium SR-11 wild-type strain (R. Mercado-Lubo, E. J. Gauger, M. P. Leatham, T. Conway, and P. S. Cohen, Infect. Immun. 76:1128-1134, 2008). Furthermore, the ability to convert succinate to fumarate appeared to be required only after serovar Typhimurium SR-11 became systemic. In the present study, evidence is presented that serovar Typhimurium SR-11 mutants that cannot convert fumarate to malate or that cannot convert malate to both oxaloacetate and pyruvate are also avirulent and protective in BALB/c mice. These results suggest that in BALB/c mice, the malate that is removed from the TCA cycle in serovar Typhimurium SR-11 for conversion to pyruvate must be replenished by succinate or one of its precursors, e.g., arginine or ornithine, which might be available in mouse phagocytes.

Palladium alpha-lipoic acid complex formulation enhances activities of Krebs cycle dehydrogenases and respiratory complexes I-IV in the heart of aged rats.

PubMed

Sudheesh, N P; Ajith, T A; Janardhanan, K K; Krishnan, C V

2009-08-01

Age-related decline in the capacity to withstand stress, such as ischemia and reperfusion, results in congestive heart failure. Though the mechanisms underlying cardiac decay are not clear, age dependent somatic damages to mitochondrial DNA (mtDNA), loss of mitochondrial function, and a resultant increase in oxidative stress in heart muscle cells may be responsible for the increased risk for cardiovascular diseases. The effect of a safe nutritional supplement, POLY-MVA, containing the active ingredient palladium alpha-lipoic acid complex, was evaluated on the activities of the Krebs cycle enzymes such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV in heart mitochondria of aged male albino rats of Wistar strain. Administration of 0.05 ml/kg of POLY-MVA (which is equivalent to 0.38 mg complexed alpha-lipoic acid/kg, p.o), once daily for 30 days, was significantly (p<0.05) effective to enhance the Krebs cycle dehydrogenases, and mitochondrial electron transport chain complexes. The unique electronic and redox properties of palladium alpha-lipoic acid complex appear to be a key to this physiological effectiveness. The results strongly suggest that this formulation might be effective to protect the aging associated risk of cardiovascular and neurodegenerative diseases.

Bundle-sheath thylakoids from NADP-malic enzyme-type C4 plants require an exogenous electron donor for enzyme light activation.

PubMed

Lavergne, D; Droux, M; Jacquot, J P; Miginiac-Maslow, M; Champigny, M L; Gadal, P

1985-10-01

Light activation of either NADP-malate dehydrogenase (EC 1.1.1.82) or fructose-1,6-bisphosphate phosphatase (EC 3.1.3.11) was assayed in a reconstituted chloroplastic, system comprising the isolated proteins of the ferredoxin-thioredoxin light-activation system and thylakoids from either mesophyll or bundle-sheath tissues of different C4 plants. While C4-plant thylakoids functionned almost equally well with C3-or C4-plant proteins, the photosyntem-II-deficient bundle-sheath thylakoids from the NADP-malic enzyme type, were unable to perform enzyme photoactivation unless supplemented with an electron donor to photosystem I. Bundle-sheath thylakoids isolated from plants showing no photosystem-II deficiency did not require such an addition. The results are discussed with respect to a possible requirement for a physiological reductant of ferredoxin for enzyme light activation in bundle-sheath, tissues.

Functional, structural and phylogenetic analysis of domains underlying the Al-sensitivity of the aluminium-activated malate/anion transporter, TaALMT1

USDA-ARS?s Scientific Manuscript database

TaALMT1 (Triticum aestivum Aluminum Activated Malate Transporter) is the founding member of a novel gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small subgroup of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (...

Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance

USDA-ARS?s Scientific Manuscript database

Aluminum (Al) activated root malate and citrate exudation plays an important role in Al tolerance in many plant species. Here, we report on the identification and characterization of AtMATE, a homolog of the recently discovered sorghum and barley Al tolerance genes, here shown to encode an Al-activ...

Metabolic consequences of limited substrate anion permeability in brown fat mitochondria from a hibernator, the golden hamster.

PubMed

Cannon, B; Bernson, V S; Nedergaard, J

1984-08-31

Brown fat mitochondria obtained from a hibernator, the golden hamster, were investigated in order to elucidate the significance of membrane permeability for metabolic functioning at different temperatures. The mitochondria were shown to have active permeases for phosphate and pyruvate, but very poorly developed permeases for di- and tricarboxylate substrate anions. This was shown with both osmotic swelling techniques and respiration-driven uptake studies. It was shown that the very limited malate permeation observed was compatible with it being a non-carrier-mediated diffusion process. The role of malate transport in supporting fatty-acid oxidation in vitro as a function of temperature was studied in detail. The results support our earlier suggestion that physiologically pyruvate carboxylase probably functions to generate oxaloacetate when high concentrations of condensing partner are needed during thermogenesis. They may also explain earlier observations that acetate was produced from palmitoyl-carnitine at low temperatures even when malate was present; this is here shown to be due to the limited malate permeability at these low temperatures. Thus, even at the body temperature of the hibernating hamster (4-5 degrees C), brown fat is probably able to combust fatty acids totally.

Cyanide degradation by Pseudomonas pseudoalcaligenes CECT5344 involves a malate:quinone oxidoreductase and an associated cyanide-insensitive electron transfer chain.

PubMed

Luque-Almagro, Victor M; Merchán, Faustino; Blasco, Rafael; Igeño, M Isabel; Martínez-Luque, Manuel; Moreno-Vivián, Conrado; Castillo, Francisco; Roldán, M Dolores

2011-03-01

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 is able to grow with cyanide as the sole nitrogen source. Membrane fractions from cells grown under cyanotrophic conditions catalysed the production of oxaloacetate from L-malate. Several enzymic activities of the tricarboxylic acid and glyoxylate cycles in association with the cyanide-insensitive respiratory pathway seem to be responsible for the oxaloacetate formation in vivo. Thus, in cyanide-grown cells, citrate synthase and isocitrate lyase activities were significantly higher than those observed with other nitrogen sources. Malate dehydrogenase activity was undetectable, but a malate:quinone oxidoreductase activity coupled to the cyanide-insensitive alternative oxidase was found in membrane fractions from cyanide-grown cells. Therefore, oxaloacetate production was linked to the cyanide-insensitive respiration in P. pseudoalcaligenes CECT5344. Cyanide and oxaloacetate reacted chemically inside the cells to produce a cyanohydrin (2-hydroxynitrile), which was further converted to ammonium. In addition to cyanide, strain CECT5344 was able to grow with several cyano derivatives, such as 2- and 3-hydroxynitriles. The specific system required for uptake and metabolization of cyanohydrins was induced by cyanide and by 2-hydroxynitriles, such as the cyanohydrins of oxaloacetate and 2-oxoglutarate.

Apparent Km of mitochondria for oxygen computed from Vmax measured in permeabilized muscle fibers is lower in water enriched in oxygen by electrolysis than injection

PubMed Central

Zoll, Joffrey; Bouitbir, Jamal; Sirvent, Pascal; Klein, Alexis; Charton, Antoine; Jimenez, Liliana; Péronnet, François R; Geny, Bernard; Richard, Ruddy

2015-01-01

Background It has been suggested that oxygen (O2) diffusion could be favored in water enriched in O2 by a new electrolytic process because of O2 trapping in water superstructures (clathrates), which could reduce the local pressure/content relationships for O2 and facilitate O2 diffusion along PO2 gradients. Materials and methods Mitochondrial respiration was compared in situ in saponin-skinned fibers isolated from the soleus muscles of Wistar rats, in solution enriched in O2 by injection or the electrolytic process 1) at an O2 concentration decreasing from 240 µmol/L to 10 µmol/L (132 mmHg to 5 mmHg), with glutamate–malate or N, N, N′, N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)–ascorbate (with antimycin A) as substrates; and 2) at increasing adenosine diphosphate (ADP) concentration with glutamate–malate as substrate. Results As expected, maximal respiration decreased with O2 concentration and, when compared to glutamate–malate, the apparent Km O2 of mitochondria for O2 was significantly lower with TMPD–ascorbate with both waters. However, when compared to the water enriched in O2 by injection, the Km O2 was significantly lower with both electron donors in water enriched in O2 by electrolysis. This was not associated with any increase in the sensitivity of mitochondria to ADP; no significant difference was observed for the Km ADP between the two waters. Conclusion In this experiment, a higher affinity of the mitochondria for O2 was observed in water enriched in O2 by electrolysis than by injection. This observation is consistent with the hypothesis that O2 diffusion can be facilitated in water enriched in O2 by the electrolytic process. PMID:26203225

Apparent Km of mitochondria for oxygen computed from Vmax measured in permeabilized muscle fibers is lower in water enriched in oxygen by electrolysis than injection.

PubMed

Zoll, Joffrey; Bouitbir, Jamal; Sirvent, Pascal; Klein, Alexis; Charton, Antoine; Jimenez, Liliana; Péronnet, François R; Geny, Bernard; Richard, Ruddy

2015-01-01

It has been suggested that oxygen (O2) diffusion could be favored in water enriched in O2 by a new electrolytic process because of O2 trapping in water superstructures (clathrates), which could reduce the local pressure/content relationships for O2 and facilitate O2 diffusion along PO2 gradients. Mitochondrial respiration was compared in situ in saponin-skinned fibers isolated from the soleus muscles of Wistar rats, in solution enriched in O2 by injection or the electrolytic process 1) at an O2 concentration decreasing from 240 µmol/L to 10 µmol/L (132 mmHg to 5 mmHg), with glutamate-malate or N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)-ascorbate (with antimycin A) as substrates; and 2) at increasing adenosine diphosphate (ADP) concentration with glutamate-malate as substrate. As expected, maximal respiration decreased with O2 concentration and, when compared to glutamate-malate, the apparent Km O2 of mitochondria for O2 was significantly lower with TMPD-ascorbate with both waters. However, when compared to the water enriched in O2 by injection, the Km O2 was significantly lower with both electron donors in water enriched in O2 by electrolysis. This was not associated with any increase in the sensitivity of mitochondria to ADP; no significant difference was observed for the Km ADP between the two waters. In this experiment, a higher affinity of the mitochondria for O2 was observed in water enriched in O2 by electrolysis than by injection. This observation is consistent with the hypothesis that O2 diffusion can be facilitated in water enriched in O2 by the electrolytic process.

NADP-Malate Dehydrogenase of Sweet Sorghum Improves Salt Tolerance of Arabidopsis thaliana.

PubMed

Guo, Yuanyuan; Song, Yushuang; Zheng, Hongxiang; Zhang, Yi; Guo, Jianrong; Sui, Na

2018-06-08

Sweet sorghum is a C 4 crop that shows high salt tolerance and high yield. NADP-malate dehydrogenase ( NADP-ME) is a crucial enzyme of the C 4 pathway. The regulatory mechanism of NADP-ME remains unclear. In this study, we isolated SbNADP-ME from sweet sorghum. The open reading frame of SbNADP-ME is 1911 bp and 637 amino acid residues. Quantitative real-time PCR analysis showed that SbNADP-ME transcription in sweet sorghum was enhanced by salt stress. The SbNADP-ME transcript level was highest under exposure to 150 mM NaCl. Arabidopsis overexpressing SbNADP-ME showed increased germination rate and root length under NaCl treatments. At the seedling stage, physiological photosynthesis parameters, chlorophyll content, PSII photochemical efficiency, and PSI oxidoreductive activity in the wild type decreased more severely than in the overexpression lines but less than in T-DNA insertion mutants under salt stress. Overexpression of SbNADP-ME in Arabidopsis may also increase osmotic adjustment and scavenging activity on DPPH and decrease membrane peroxidation. These results suggest that SbNADP-ME overexpression in Arabidopsis increases salt tolerance and alleviates PSII and PSI photoinhibition under salt stress by improving photosynthetic capacity.

Lipstick dermatitis due to C18 aliphatic compounds.

PubMed

Hayakawa, R; Matsunaga, K; Suzuki, M; Arima, Y; Ohkido, Y

1987-04-01

An 18-year-old girl developed cheilitis. She had a past history of lip cream dermatitis, but the cause was not found. Patch tests with 2 lipsticks were strongly positive. Tests with the ingredients were positive to 2 aliphatic compounds, glyceryl diisostearate and diisostearyl malate. Impurities in the materials were suspected as the cause. Analysis by gas chromatography detected 3 chemicals in glyceryl diisostearate and 1 in diisostearyl malate as impurities. Patch testing with the impurities and glyceryl monoisostearate 0.01% pet in glyceryl diisostearate and isostearyl alcohol 0.25% pet in diisostearyl malate were strongly positive. The characteristics common to the 2 chemicals were liquidity at room temperature, branched C18 aliphatic compound and primary alcohol. Chemicals lacking any of the above 3 features did not react.

Combined metabolomic and correlation networks analyses reveal fumarase insufficiency altered amino acid metabolism.

PubMed

Hou, Entai; Li, Xian; Liu, Zerong; Zhang, Fuchang; Tian, Zhongmin

2018-04-01

Fumarase catalyzes the interconversion of fumarate and l-malate in the tricarboxylic acid cycle. Fumarase insufficiencies were associated with increased levels of fumarate, decreased levels of malate and exacerbated salt-induced hypertension. To gain insights into the metabolism profiles induced by fumarase insufficiency and identify key regulatory metabolites, we applied a GC-MS based metabolomics platform coupled with a network approach to analyze fumarase insufficient human umbilical vein endothelial cells (HUVEC) and negative controls. A total of 24 altered metabolites involved in seven metabolic pathways were identified as significantly altered, and enriched for the biological module of amino acids metabolism. In addition, Pearson correlation network analysis revealed that fumaric acid, l-malic acid, l-aspartic acid, glycine and l-glutamic acid were hub metabolites according to Pagerank based on their three centrality indices. Alanine aminotransferase and glutamate dehydrogenase activities increased significantly in fumarase deficiency HUVEC. These results confirmed that fumarase insufficiency altered amino acid metabolism. The combination of metabolomics and network methods would provide another perspective on expounding the molecular mechanism at metabolomics level. Copyright © 2017 John Wiley & Sons, Ltd.

Low-molecular-weight poly(alpha-methyl beta,L-malate) of microbial origin: synthesis and crystallization.

PubMed

Fernández, Carlos E; Mancera, Manuel; Holler, Eggehard; Bou, Jordi J; Galbis, Juan A; Muñoz-Guerra, Sebastián

2005-02-23

Low-molecular-weight poly(alpha-methyl beta,L-malate) made of approximately 25-30 units was prepared from microbial poly(beta,L-malic acid) by treatment with diazomethane. The thermal characterization of the polymalate methyl ester was carried out and its crystalline structure was preliminary examined. Its ability to crystallize both from solution and from the melt was comparatively evaluated.

A domain-based approach for analyzing the function of aluminum-activated malate transporters from wheat (Triticum aestivum) and Arabidopsis thaliana in Xenopus oocytes.

PubMed

Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Furuichi, Takuya; Yamamoto, Yoko

2014-12-01

Wheat and Arabidopsis plants respond to aluminum (Al) ions by releasing malate from their root apices via Al-activated malate transporter. Malate anions bind with the toxic Al ions and contribute to the Al tolerance of these species. The genes encoding the transporters in wheat and Arabidopsis, TaALMT1 and AtALMT1, respectively, were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the two-electrode voltage clamp system. The Al-activated currents generated by malate efflux were detected for TaALMT1 but not for AtALMT1. Chimeric proteins were generated by swapping the N- and C-terminal halves of TaALMT1 and AtALMT1 (Ta::At and At::Ta). When these chimeras were characterized in oocytes, Al-activated malate efflux was detected for the Ta::At chimera but not for At::Ta, suggesting that the N-terminal half of TaALMT1 is necessary for function in oocytes. An additional chimera, Ta(48)::At, generated by swapping 17 residues from the N-terminus of AtALMT1 with the equivalent 48 residues from TaALMT1, was sufficient to support transport activity. This 48 residue region includes a helical region with a putative transmembrane domain which is absent in AtALMT1. The deletion of this domain from Ta(48)::At led to the complete loss of transport activity. Furthermore, truncations and a deletion at the C-terminal end of TaALMT1 indicated that a putative helical structure in this region was also required for transport function. This study provides insights into the structure-function relationships of Al-activated ALMT proteins by identifying specific domains on the N- and C-termini of TaALMT1 that are critical for basal transport function and Al responsiveness in oocytes. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boernke, W. E.; Millard, C. S.; Stevens, P. W.

1995-09-10

Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the nativemore » enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant malate dehydrogenase. However, when expressed in a strain of E. coli unable to ferment glucose, the mutant enzyme restored growth and produced lactic acid as the sole fermentation product.« less

Peroxisomal Malate Dehydrogenase Is Not Essential for Photorespiration in Arabidopsis But Its Absence Causes an Increase in the Stoichiometry of Photorespiratory CO2 Release1[W][OA

PubMed Central

Cousins, Asaph B.; Pracharoenwattana, Itsara; Zhou, Wenxu; Smith, Steven M.; Badger, Murray R.

2008-01-01

Peroxisomes are important for recycling carbon and nitrogen that would otherwise be lost during photorespiration. The reduction of hydroxypyruvate to glycerate catalyzed by hydroxypyruvate reductase (HPR) in the peroxisomes is thought to be facilitated by the production of NADH by peroxisomal malate dehydrogenase (PMDH). PMDH, which is encoded by two genes in Arabidopsis (Arabidopsis thaliana), reduces NAD+ to NADH via the oxidation of malate supplied from the cytoplasm to oxaloacetate. A double mutant lacking the expression of both PMDH genes was viable in air and had rates of photosynthesis only slightly lower than in the wild type. This is in contrast to other photorespiratory mutants, which have severely reduced rates of photosynthesis and require high CO2 to grow. The pmdh mutant had a higher O2-dependent CO2 compensation point than the wild type, implying that either Rubisco specificity had changed or that the rate of CO2 released per Rubisco oxygenation was increased in the pmdh plants. Rates of gross O2 evolution and uptake were similar in the pmdh and wild-type plants, indicating that chloroplast linear electron transport and photorespiratory O2 uptake were similar between genotypes. The CO2 postillumination burst and the rate of CO2 released during photorespiration were both greater in the pmdh mutant compared with the wild type, suggesting that the ratio of photorespiratory CO2 release to Rubisco oxygenation was altered in the pmdh mutant. Without PMDH in the peroxisome, the CO2 released per Rubisco oxygenation reaction can be increased by over 50%. In summary, PMDH is essential for maintaining optimal rates of photorespiration in air; however, in its absence, significant rates of photorespiration are still possible, indicating that there are additional mechanisms for supplying reductant to the peroxisomal HPR reaction or that the HPR reaction is altogether circumvented. PMID:18685043

Profiling Taste and Aroma Compound Metabolism during Apricot Fruit Development and Ripening

PubMed Central

Xi, Wanpeng; Zheng, Huiwen; Zhang, Qiuyun; Li, Wenhui

2016-01-01

Sugars, organic acids and volatiles of apricot were determined by HPLC and GC-MS during fruit development and ripening, and the key taste and aroma components were identified by integrating flavor compound contents with consumers’ evaluation. Sucrose and glucose were the major sugars in apricot fruit. The contents of all sugars increased rapidly, and the accumulation pattern of sugars converted from glucose-predominated to sucrose-predominated during fruit development and ripening. Sucrose synthase (SS), sorbitol oxidase (SO) and sorbitol dehydrogenase (SDH) are under tight developmental control and they might play important roles in sugar accumulation. Almost all organic acids identified increased during early development and then decrease rapidly. During early development, fruit mainly accumulated quinate and malate, with the increase of citrate after maturation, and quinate, malate and citrate were the predominant organic acids at the ripening stage. The odor activity values (OAV) of aroma volatiles showed that 18 aroma compounds were the characteristic components of apricot fruit. Aldehydes and terpenes decreased significantly during the whole development period, whereas lactones and apocarotenoids significantly increased with fruit ripening. The partial least squares regression (PLSR) results revealed that β-ionone, γ-decalactone, sucrose and citrate are the key characteristic flavor factors contributing to consumer acceptance. Carotenoid cleavage dioxygenases (CCD) may be involved in β-ionone formation in apricot fruit. PMID:27347931

Malate as a key carbon source of leaf dark-respired CO2 across different environmental conditions in potato plants

PubMed Central

Lehmann, Marco M.; Rinne, Katja T.; Blessing, Carola; Siegwolf, Rolf T. W.; Buchmann, Nina; Werner, Roland A.

2015-01-01

Dissimilation of carbon sources during plant respiration in support of metabolic processes results in the continuous release of CO2. The carbon isotopic composition of leaf dark-respired CO2 (i.e. δ 13 C R) shows daily enrichments up to 14.8‰ under different environmental conditions. However, the reasons for this 13C enrichment in leaf dark-respired CO2 are not fully understood, since daily changes in δ13C of putative leaf respiratory carbon sources (δ 13 C RS) are not yet clear. Thus, we exposed potato plants (Solanum tuberosum) to different temperature and soil moisture treatments. We determined δ 13 C R with an in-tube incubation technique and δ 13 C RS with compound-specific isotope analysis during a daily cycle. The highest δ 13 C RS values were found in the organic acid malate under different environmental conditions, showing less negative values compared to δ 13 C R (up to 5.2‰) and compared to δ 13 C RS of soluble carbohydrates, citrate and starch (up to 8.8‰). Moreover, linear relationships between δ 13 C R and δ 13 C RS among different putative carbon sources were strongest for malate during daytime (r2=0.69, P≤0.001) and nighttime (r2=0.36, P≤0.001) under all environmental conditions. A multiple linear regression analysis revealed δ 13 C RS of malate as the most important carbon source influencing δ 13 C R. Thus, our results strongly indicate malate as a key carbon source of 13C enriched dark-respired CO2 in potato plants, probably driven by an anapleurotic flux replenishing intermediates of the Krebs cycle. PMID:26139821

Glycosomal and mitochondrial malate dehydrogenases in epimastigotes of Trypanosoma cruzi.

PubMed

Cannata, J J; Cazzulo, J J

1984-04-01

The degradation of glucose by Trypanosoma cruzi leads to the excretion of succinate. Malate dehydrogenase (MDH) participates in this process by reducing to malate the oxaloacetate synthesized by the glycosomal enzyme, phosphoenolpyruvate carboxykinase. The best coupling for these two sequential reactions would be attained if both enzymes were placed in the same subcellular compartment. The intracellular distribution of the MDH activity in epimastigotes of T. cruzi was studied by two methods. Selective disruption of cellular membranes with increasing concentrations of digitonin, indicated that trypanosomal MDH is particulate. Isopycnic centrifugation in a sucrose gradient of a large granule fraction, obtained by grinding the cells with silicon carbide, showed the presence of two MDH activities: one banding together with the glycosomal marker phosphoenolpyruvate carboxykinase, the other with the mitochondrial marker citrate synthase. Isoelectrofocusing of cell-free extracts led to the separation of two enzyme forms, with pI values of about 3.5 (MDHa) and 9.4 (MDHb). These forms had similar molecular weights (approx. 60 000) and apparent Km values, but showed a small but consistent difference in their pH optima (9.23 for MDHa and 9.05 for MDHb), and in their activation by inorganic phosphate (apparent Ka values of 33 mM and 87 mM, for MDHa and MDHb, respectively). Determination of the pH optima of the enzyme forms separated by isopycnic centrifugation suggests that the glycosomal enzyme form is MDHa, and the mitochondrial one is MDHb.

Characterization of the soybean GmALMT family genes and the function of GmALMT5 in response to phosphate starvation.

PubMed

Peng, Wenting; Wu, Weiwei; Peng, Junchu; Li, Jiaojiao; Lin, Yan; Wang, Yanan; Tian, Jiang; Sun, Lili; Liang, Cuiyue; Liao, Hong

2018-03-01

A potential mechanism to enhance utilization of sparingly soluble forms of phosphorus (P) is the root secretion of malate, which is mainly mediated by the ALMT gene family in plants. In this study, a total of 34 GmALMT genes were identified in the soybean genome. Expression patterns diverged considerably among GmALMTs in response to phosphate (Pi) starvation in leaves, roots and flowers, with expression altered by P availability in 26 of the 34 GmALMTs. One root-specific GmALMT whose expression was significantly enhanced by Pi-starvation, GmALMT5, was studied in more detail to determine its possible role in soybean P nutrition. Analysis of GmALMT5 tissue expression patterns, subcellular localization, and malate exudation from transgenic soybean hairy roots overexpressing GmALMT5, demonstrated that GmALMT5 is a plasma membrane protein that mediates malate efflux from roots. Furthermore, both growth and P content of transgenic Arabidopsis overexpressing GmALMT5 were significantly increased when sparingly soluble Ca-P was used as the external P source. Taken together, these results indicate that members of the soybean GmALMT gene family exhibit diverse responses to Pi starvation. One member of this family, GmALMT5, might contribute to soybean P efficiency by enhancing utilization of sparingly soluble P sources under P limited conditions. © 2017 Institute of Botany, Chinese Academy of Sciences.

The varied functions of aluminium-activated malate transporters–much more than aluminium resistance

PubMed Central

Palmer, Antony J.; Baker, Alison; Muench, Stephen P.

2016-01-01

The ALMT (aluminium-activated malate transporter) family comprises a functionally diverse but structurally similar group of ion channels. They are found ubiquitously in plant species, expressed throughout different tissues, and located in either the plasma membrane or tonoplast. The first family member identified was TaALMT1, discovered in wheat root tips, which was found to be involved in aluminium resistance by means of malate exudation into the soil. However, since this discovery other family members have been shown to have many other functions such as roles in stomatal opening, general anionic homoeostasis, and in economically valuable traits such as fruit flavour. Recent evidence has also shown that ALMT proteins can act as key molecular actors in GABA (γ-aminobutyric acid) signalling, the first evidence that GABA can act as a signal transducer in plants. PMID:27284052

[Enzyme activity in the subcellular fractions of the liver of rats following a flight on board the Kosmos-1129 biosatellite].

PubMed

Tigranian, R A; Vetrova, E G; Abraham, S; Lin, C; Klein, H

1983-01-01

The activities of malate, isocitrate, and lactate dehydrogenases were measured in the liver mitochondrial and cytoplasmatic fractions of rats flown for 18.5 days onboard Cosmos-1129. The activities of the oxidative enzymes, malate and isocitrate dehydrogenases, in the mitochondrial fraction and those of the glycolytic enzyme, lactate dehydrogenase, in the cytoplasmatic fraction were found to decrease.

The glyoxylate pathway contributes to enhanced extracellular electron transfer in yeast-based biofuel cell.

PubMed

Hubenova, Yolina; Hubenova, Eleonora; Slavcheva, Evelina; Mitov, Mario

2017-08-01

This study provides a new insight into our understanding of yeast response to starvation conditions (sole acetate as carbon source) and applied polarization and offers important information about the role of the glyoxylate cycle in the carbohydrate synthesis and extracellular charge transfer processes in biofuel cells. The biosynthetic capabilities of yeast C. melibiosica 2491 and the up/down-regulation of the glyoxylate cycle are evaluated by modifying the cellular metabolism by feedback inhibition or carbohydrate presence and establishing the malate dehydrogenase activity and carbohydrate content together with the electric charge passed through bioelectrochemical system. 10mM malate leads to a decrease of the produced quantity of electricity with ca. 55%. At the same time, 24-times lower intracellular malate dehydrogenase activity is established. At polarization conditions the glyoxylate pathway is up-regulated and huge amount of malate is intra-converted into oxaloacetate. The yeasts are able to synthesize carbohydrates from acetate and a part of them is used for the electricity generation. It is recognized that the enhanced charge transfer in acetate fed yeast-based biofuel cell is implemented by secreted endogenous mediator and changes in the cellular surface redox activity depending on the addition of carbohydrate in the medium. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterisation of the two malate dehydrogenases from Phytomonas sp. Purification of the glycosomal isoenzyme.

PubMed

Uttaro, A D; Opperdoes, F R

1997-10-01

Two NAD(H)-dependent malate dehydrogenase (MDH) isoenzymes were detected in Phytomonas isolated from the lactiferous tubes of Euphorbia characias. The total specific activity in crude extracts using oxaloacetate as substrate was 3.3 U mg-1 of protein. The two isoenzymes had isoelectric points of 6.0 and 7.2, respectively. The acidic isoform represented 80% of the total activity in the cell and was present in the glycosome. It was purified to homogeneity by a method involving hydrophobic interaction chromatography on Phenyl-Sepharose followed by ionic exchange on CM-Sepharose and affinity chromatography on Blue-Sepharose. The purified glycosomal MDH is a homodimeric protein with a subunit molecular mass of 37 kDa and it has a low substrate specificity, since it was able to reduce both aromatic and aliphatic alpha-ketoacids as substrate including oxaloacetate, phenyl pyruvate, alpha-keto iso-caproate and pyruvate. The apparent K(m)s for oxaloacetate and NADH were 166 and 270 microM, respectively and for L-malate and NAD+, 3000 and 246 microM, respectively. The basic isoform was present in the mitochondrion. It has a high substrate specificity and an apparent K(m) of 132 and 63 microM for oxaloacetate and NADH, respectively, and of 450 and 91 microM, respectively, with L-malate and NAD+.

Interactions of C 4 Subtype Metabolic Activities and Transport in Maize Are Revealed through the Characterization of DCT2 Mutants

DOE PAGES

Weissmann, Sarit; Ma, Fangfang; Furuyama, Koki; ...

2016-01-26

C 4 photosynthesis in grasses requires the coordinated movement of metabolites through two specialized leaf cell types, mesophyll (M) and bundle sheath (BS), to concentrate CO 2 around Rubisco. Despite the importance of transporters in this process, few have been identified or rigorously characterized. In maize (Zea mays), DCT2 has been proposed to function as a plastid-localizedmalate transporter and is preferentially expressed in BS cells. Here, we characterized the role of DCT2 in maize leaves using Activator-tagged mutant alleles. Our results indicate that DCT2 enables the transport of malate into the BS chloroplast. Isotopic labeling experiments show that the lossmore » of DCT2 results in markedly different metabolic network operation and dramatically reduced biomass production. In the absence of a functioning malate shuttle, dct2 lines survive through the enhanced use of the phosphoenolpyruvate carboxykinase carbon shuttle pathway that in wild-type maize accounts for ;25% of the photosynthetic activity. The results emphasize the importance of malate transport during C 4 photosynthesis, define the role of a primary malate transporter in BS cells, and support a model for carbon exchange between BS and M cells in maize.« less

Microbial reduction of Fe(III) in acidic sediments: isolation of Acidiphilium cryptum JF-5 capable of coupling the reduction of Fe(III) to the oxidation of glucose.

PubMed

Küsel, K; Dorsch, T; Acker, G; Stackebrandt, E

1999-08-01

To evaluate the microbial populations involved in the reduction of Fe(III) in an acidic, iron-rich sediment, the anaerobic flow of supplemental carbon and reductant was evaluated in sediment microcosms at the in situ temperature of 12 degrees C. Supplemental glucose and cellobiose stimulated the formation of Fe(II); 42 and 21% of the reducing equivalents that were theoretically obtained from glucose and cellobiose, respectively, were recovered in Fe(II). Likewise, supplemental H(2) was consumed by acidic sediments and yielded additional amounts of Fe(II) in a ratio of approximately 1:2. In contrast, supplemental lactate did not stimulate the formation of Fe(II). Supplemental acetate was not consumed and inhibited the formation of Fe(II). Most-probable-number estimates demonstrated that glucose-utilizing acidophilic Fe(III)-reducing bacteria approximated to 1% of the total direct counts of 4', 6-diamidino-2-phenylindole-stained bacteria. From the highest growth-positive dilution of the most-probable-number series at pH 2. 3 supplemented with glucose, an isolate, JF-5, that could dissimilate Fe(III) was obtained. JF-5 was an acidophilic, gram-negative, facultative anaerobe that completely oxidized the following substrates via the dissimilation of Fe(III): glucose, fructose, xylose, ethanol, glycerol, malate, glutamate, fumarate, citrate, succinate, and H(2). Growth and the reduction of Fe(III) did not occur in the presence of acetate. Cells of JF-5 grown under Fe(III)-reducing conditions formed blebs, i.e., protrusions that were still in contact with the cytoplasmic membrane. Analysis of the 16S rRNA gene sequence of JF-5 demonstrated that it was closely related to an Australian isolate of Acidiphilium cryptum (99.6% sequence similarity), an organism not previously shown to couple the complete oxidation of sugars to the reduction of Fe(III). These collective results indicate that the in situ reduction of Fe(III) in acidic sediments can be mediated by heterotrophic Acidiphilium species that are capable of coupling the reduction of Fe(III) to the complete oxidation of a large variety of substrates including glucose and H(2).

A Concise History of the U.S. Army Signal Corps

DTIC Science & Technology

1991-02-01

key information in defeating the Spanish. Signal Corps units participated in the capture of Manila and the assault on Fort San Antonio and Fort Malate ...advancing infantry’s position. Other signalmen rolled in wire while Sergeant Harry Chadwick photographed the events. The Americans took Fort Malate ...with the Mekong Delta’s monsoon-ridden soil , a poor foundation for communications equipment. Like signalmen of the past, they adapted to local

The varied functions of aluminium-activated malate transporters-much more than aluminium resistance.

PubMed

Palmer, Antony J; Baker, Alison; Muench, Stephen P

2016-06-15

The ALMT (aluminium-activated malate transporter) family comprises a functionally diverse but structurally similar group of ion channels. They are found ubiquitously in plant species, expressed throughout different tissues, and located in either the plasma membrane or tonoplast. The first family member identified was TaALMT1, discovered in wheat root tips, which was found to be involved in aluminium resistance by means of malate exudation into the soil. However, since this discovery other family members have been shown to have many other functions such as roles in stomatal opening, general anionic homoeostasis, and in economically valuable traits such as fruit flavour. Recent evidence has also shown that ALMT proteins can act as key molecular actors in GABA (γ-aminobutyric acid) signalling, the first evidence that GABA can act as a signal transducer in plants. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Malic enzyme: Tritium isotope effects with alternative dinucleotide substrates and divalent metal ions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karsten, W.E.; Harris, B.G.; Cook, P.F.

1992-01-01

The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion dependent oxidative decarboxylation of L-malate to yield pyruvate, carbon dioxide and NADH. Multiple isotope effect studies suggest a stepwise chemical mechanism with hydride transfer from L-malate to NAD occurring first to form oxalacetate, followed by decarboxylation. Utilizing L-malate-2-T, tritium V/K isotope effects have been determined for the hydride transfer step using a variety of alternative dinucleotide substrates and divalent metal ions. Combination of these data with deuterium isotope effects data and previously determined [sup 13]C isotope effects has allowed the calculation of intrinsic isotope effects for the malic enzymemore » catalyzed reaction. The identity of both the dinucleotide substrate and divalent metal ion has an effect of the size of the intrinsic isotope effect for hydride transfer.« less

Silent information regulator 1 (SIRT1) ameliorates liver fibrosis via promoting activated stellate cell apoptosis and reversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Yuting, E-mail: wuyuting1302@sina.com; Liu, Xuejiao; Zhou, Qun

SIRT1 (silent information regulator 1), a conserved NAD +-dependent histone deacetylase, is closely related with various biological processes. Moreover, the important role of SIRT1 in alcoholic liver disease, nonalcoholic fatty liver and HCC had been widely reported. Recently, a novel role of SIRT1 was uncovered in organ fibrosis diseases. Here, we investigated the inhibitory effect of SIRT1 in liver fibrogenesis. SIRT1 protein was dramatically decreased in CCl4-treated mice livers. Stimulation of LX-2 cells with TGF-β1 also resulted in a significant suppression of SIRT1 protein. Nevertheless, TGF-β1-induced LX-2 cell activation was inhibited by SIRT1 plasmid, and this was accompanied by up-regulationmore » of cell apoptosis-related proteins. Overexpression of SIRT1 also attenuated TGF-β1-induced expression of myofibroblast markers α-SMA and COL1a. However, the important characteristic of the recovery of liver fibrosis is not only the apoptosis of activated stellate cells but also the reversal of the myofibroblast-like phenotype to a quiescent-like phenotype. Restoration of SIRT1 protein was observed in the in vivo spontaneously liver fibrosis reversion model and in vitro MDI (isobutylmethylxanthine, dexamethasone, and insulin)-induced reversed stellate cells, and forced expression of SIRT1 also promoted the reversal of activated stellate cells. Furthermore, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was increased in liver fibrosis. RNAi-mediated suppression of MALAT1 resulted in a decrease of myofibroblast markers and restoration of SIRT1 protein. These observations suggested that SIRT1 contributed to apoptosis and reversion of activated LX-2 cells and SIRT1 might be regulated by MALAT1 in liver fibrosis. Therefore, SIRT1 could be considered as a valuable therapeutic target for translational studies of liver fibrosis. - Highlights: • This is the first report of SIRT1 expression and function in liver fibrogenesis and reversion. • Aberrant expression of SIRT1 might just occur at a post-transcriptional level. • LncRNA MALAT1 might be responsible for the changes of SIRT1 in liver fibrosis.« less

Path of carbon flow during NO/sub 3//sup -/-induced photosynthetic suppression in N-limited Selenastrum minutum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elrifi, I.R.; Turpin, D.H.

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In /sup 14/CO/sub 2/ pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4more » position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 ..mu..moles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 ..mu..moles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation.« less

The Path of Carbon Flow during NO3−-Induced Photosynthetic Suppression in N-Limited Selenastrum minutum1

PubMed Central

Elrifi, Ivor R.; Turpin, David H.

1987-01-01

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In 14CO2 pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4 position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 μmoles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 μmoles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation (IR Elrifi, DH Turpin 1986 Plant Physiol 81: 273-279). PMID:16665223

The Path of Carbon Flow during NO(3)-Induced Photosynthetic Suppression in N-Limited Selenastrum minutum.

PubMed

Elrifi, I R; Turpin, D H

1987-01-01

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In (14)CO(2) pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4 position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 mumoles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 mumoles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation (IR Elrifi, DH Turpin 1986 Plant Physiol 81: 273-279).

Comparative Analysis of the Symbiotic Efficiency of Medicago truncatula and Medicago sativa under Phosphorus Deficiency

PubMed Central

Sulieman, Saad; Schulze, Joachim; Tran, Lam-Son Phan

2013-01-01

Phosphorus (P)-deficiency is a major abiotic stress that limits legume growth in many types of soils. The relationship between Medicago and Sinorhizobium, is known to be affected by different environmental conditions. Recent reports have shown that, in combination with S. meliloti 2011, Medicago truncatula had a lower symbiotic efficiency than Medicago sativa. However, little is known about how Medicago–Sinorhizobium is affected by P-deficiency at the whole-plant level. The objective of the present study was to compare and characterize the symbiotic efficiency of N2 fixation of M. truncatula and M. sativa grown in sand under P-limitation. Under this condition, M. truncatula exhibited a significantly higher rate of N2 fixation. The specific activity of the nodules was much higher in M. truncatula in comparison to M. sativa, partially as a result of an increase in electron allocation to N2versus H+. Although the main organic acid, succinate, exhibited a strong tendency to decrease under P-deficiency, the more efficient symbiotic ability observed in M. truncatula coincided with an apparent increase in the content of malate in its nodules. Our results indicate that the higher efficiency of the M. truncatula symbiotic system is related to the ability to increase malate content under limited P-conditions. PMID:23459233

Effect of water and nitrogen additions on free-living nitrogen fixer populations in desert grass root zones.

PubMed Central

Herman, R P; Provencio, K R; Torrez, R J; Seager, G M

1993-01-01

In this study we measured changes in population levels of free-living N2-fixing bacteria in the root zones of potted Bouteloua eriopoda and Sporobolus flexuosus plants as well as the photosynthetic indices of the plants in response to added nitrogen, added water, and added water plus nitrogen treatments. In addition, N2 fixer population changes in response to added carbon source and nitrogen were measured in plant-free soil columns. There were significant increases in the numbers of N2 fixers associated with both plant species in the water and the water plus nitrogen treatments. Both treatments increased the photosynthetic index, suggesting that plant exudates were driving N2 fixer population changes. Population increases were greatest in the water plus nitrogen treatments, indicating that added nitrogen was synergistic with added water and suggesting that nitrogen addition spared bacteria the metabolic cost of N2 fixation, allowing greater reproduction. Plant-free column studies demonstrated a synergistic carbon-nitrogen effect when carbon levels were limiting (low malate addition) but not when carbon was abundant (high malate), further supporting this hypothesis. The results of this study indicate the presence of N2 fixer populations which interact with plants and which may play a role in the nitrogen balance of desert grasslands. PMID:8215373

[Effect of salt stress on respiration metabolism in higher plants].

PubMed

Mittova, V O; Igamberdiev, A U

2000-01-01

We studied the activity of NADP-dependent isocitrate dehydrogenase, malate dehydrogenase, succinate dehydrogenase, catalase, and peroxidase as well as the rate of 14CO2 release after introduction of labeled substrates for glycolysis and citrate acid cycle within 24 h after salt stress (1% NaCl) in 10-14 days old germinants of wheat (Triticum aestivum L.) and maize (Zea mays L.) as well as thallus of small duckweed (Wolffia arrhiza (L.) Hork ex Wimmer). Oscillations in the enzymes activity with 4-6 h period have been revealed under stress conditions. Activity of glycolysis decreased in wheat and maize and increased in duckweed under the influence of stress stimulus. Six hours after NaCl action decarboxylation of exogenous citrate and succinate was enhanced in all three plants while the rate of exogenous malate decarboxylation was decreased. We conclude that adaptation of higher plans to salinization is accompanied by rearrangements in oxidative metabolism reflected by oscillations in activity of the enzymes involved in oxidative metabolism.

Effect of caffeine on the expression pattern of water-soluble proteins in rice (Oryza sativa) seedlings.

PubMed

Deng, Wei-Wei; Sasamoto, Hamako; Ashihara, Hiroshi

2015-05-01

It has been suggested that caffeine acts as an allelochemical which influences the germination and growth of plants. The effect of caffeine on the expression profiles of proteins was investigated in shoot-root axes of rice (Oryza sativa) seedlings. Two-dimensional difference gel electrophoresis combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry was employed for the separation and identification of proteins. The results indicated that amounts of 51 protein spots were reduced and 14 were increased by treatment with 1 mM caffeine. Twelve rice seedling proteins were identified. Down-regulated proteins were β-tubulin, sucrose synthase, glyceraldehyde-3-phosphate dehydrogenase, reversibly glycosylated polypeptide/α-1,4-glucan protein synthase and cytoplasmic malate dehydrogenase. In contrast, up-regulated proteins were alanyl-aminopeptidase, acetyl-CoA carboxylase, adenine phosphoribosyltransferase, NAD-malate dehydrogenase, ornithine carbamoyltransferase, glucose-6-phosphate isomerase and nuclear RNA binding protein. Possible alternation of metabolism caused by caffeine is discussed with the protein expression data.

Transport of Phosphoenolpyruvate by Chloroplasts from Mesembryanthemum crystallinum L. Exhibiting Crassulacean Acid Metabolism 1

PubMed Central

Neuhaus, H. Ekkehard; Holtum, Joseph A. M.; Latzko, Erwin

1988-01-01

Chloroplasts from CAM-Mesembryanthemum crystallinum can transport phosphoenolpyruvate (PEP) across the envelope. The initial velocities of PEP uptake in the dark at 4°C exhibited saturation kinetics with increasing external PEP concentration. PEP uptake had a Vmax of 6.46 (±0.05) micromoles per milligram chlorophyll per hour and an apparent Kmpep of 0.148 (±0.004) millimolar. The uptake was competitively inhibited by Pi (apparent Ki = 0.19 millimolar), by glycerate 3-phosphate (apparent Ki = 0.13 millimolar), and by dihydroxyacetone phosphate, but malate and pyruvate were without effect. The chloroplasts were able to synthesize PEP when presented with pyruvate. PEP synthesis was light dependent. The prolonged synthesis and export of PEP from the chloroplasts required the presence of Pi or glycerate 3-phosphate in the external medium. It is suggested that the transport of pyruvate and PEP across the chloroplasts envelope is required during the gluconeogenic conversion of carbon from malate to storage carbohydrate in the light. PMID:16666128

A new ETV6-NTRK3 cell line model reveals MALAT1 as a novel therapeutic target - a short report.

PubMed

Chen, Suning; Nagel, Stefan; Schneider, Bjoern; Dai, Haiping; Geffers, Robert; Kaufmann, Maren; Meyer, Corinna; Pommerenke, Claudia; Thress, Kenneth S; Li, Jiao; Quentmeier, Hilmar; Drexler, Hans G; MacLeod, Roderick A F

2018-02-01

Previously, the chromosomal translocation t(12;15)(p13;q25) has been found to recurrently occur in both solid tumors and leukemias. This translocation leads to ETV6-NTRK3 (EN) gene fusions resulting in ectopic expression of the NTRK3 neurotropic tyrosine receptor kinase moiety as well as oligomerization through the donated ETV6-sterile alpha motif domain. As yet, no in vitro cell line model carrying this anomaly is available. Here we genetically characterized the acute promyelocytic leukemia (APL) cell line AP-1060 and, by doing so, revealed the presence of a t(12;15)(p13;q25). Subsequently, we evaluated its suitability as a model for this important clinical entity. Spectral karyotyping, fluorescence in situ hybridization (FISH), and genomic and transcriptomic microarray-based profiling were used to screen for the presence of EN fusions. qRT-PCR was used for quantitative expression analyses. Responses to AZ-23 (NTRK) and wortmannin (PI3K) inhibitors, as well as to arsenic trioxide (ATO), were assessed using colorimetric assays. An AZ-23 microarray screen was used to define the EN targetome, which was parsed bioinformatically. MAPK1 and MALAT1 activation were assayed using Western blotting and RNA-FISH, respectively, whereas an AML patient cohort was used to assess the clinical occurrence of MALAT1 activation. An EN fusion was detected in AP1060 cells which, accordingly, turned out to be hypersensitive to AZ-23. We also found that AZ-23 can potentiate the effect of ATO and inhibit the phosphorylation of its canonical target MAPK1. The AZ-23 microarray screen highlighted a novel EN target, MALAT1, which also proved sensitive to wortmannin. Finally, we found that MALAT1 was massively up-regulated in a subset of AML patients. From our data we conclude that AP-1060 may serve as a first publicly available preclinical model for EN. In addition, we conclude that these EN-positive cells are sensitive to the NTRK inhibitor AZ-23 and that this inhibitor may potentiate the therapeutic efficacy of ATO. Our data also highlight a novel AML EN target, MALAT1, which was so far only conspicuous in solid tumors.

Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface.

PubMed

Bjørk, Alexandra; Dalhus, Bjørn; Mantzilas, Dimitrios; Eijsink, Vincent G H; Sirevåg, Reidun

2003-12-05

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers. To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions. The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg. C in apparent melting temperature compared to the wild-type. The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact. Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity. The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface.

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme.

PubMed

Chou, W Y; Chang, H P; Huang, C H; Kuo, C C; Tong, L; Chang, G G

2000-02-01

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

In vitro evaluation of the effect of botanical formulations used in the control of Aedes aegypti L. (Diptera: Culicidae) on liver enzymes.

PubMed

Porto, Karla Rejane de Andrade; Motti, Priscilla Rezende; Machado, Alexandre Alves; Roel, Antonia Railda

2016-01-01

Dengue fever is a viral disease transmitted by the Aedes aegypti Linn. (1792) (Diptera: Culicidae) mosquito, which is endemic in several regions of Brazil. Alternative methods for the control of the vector include botanical insecticides, which offer advantages such as lower environmental contamination levels and less likelihood of resistant populations. Thus, in this study, the ability of botanical insecticide formulations to inhibit the activity of the liver enzymes serum cholinesterase and malate dehydrogenase was evaluated. Inhibition profiles were assessed using in vitro assays for cholinesterase and malate dehydrogenase activity and quantitated by ultraviolet-visible spectroscopy at 410nm to 340nm. Insecticide products formulated from cashew nutshell liquid [A] and ricinoleic acid [B] showed cholinesterase activity levels of 6.26IU/mL and 6.61IU/mL, respectively, while the control level for cholinesterase was 5-12IU/mL. The products did not affect the level of 0.44IU/mL established for malate dehydrogenase, as the levels produced by [A] and [B] were 0.43IU/mL and 0.45IU/mL, respectively. Our findings show that in vitro testing of the formulated products at concentrations lethal to A. aegypti did not affect the activity of cholinesterase and malate dehydrogenase, indicating the safety of these products.

Evaluation of fluorescence in situ hybridization techniques to study long non-coding RNA expression in cultured cells

PubMed Central

Soares, Ricardo J; Maglieri, Giulia; Gutschner, Tony; Lund, Anders H; Nielsen, Boye S

2018-01-01

Abstract Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system. PMID:29059327

Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Se Jeong; Gu, Dong Ryun; Center for Metabolic Function Regulation

2016-06-17

Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reducedmore » following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.« less

Role of the mitochondrial metabolism of pyruvate on the regulation of ketogenesis in rat hepatocytes.

PubMed

Demaugre, F; Buc, H; Girard, J; Leroux, J P

1983-01-01

In hepatocytes isolated from fed rats the inhibition of lipogenesis (-80%) by 5-tetradecyloxy-2-furoate (an inhibitor of acetylCoA carboxylase) and alpha-cyano-3-hydroxycinnamate (an inhibitor of pyruvate entry into mitochondria) increases the oxidation of 0.35 mM oleate respectively by 70% and 90%. 5-tetradecyloxy-2-furoate increases ketone body production from oleate only by 30% and has no effect on ketogenesis from octanoate, whereas alpha-cyano-3-hydroxycinnamate mimics the effects of fasting on ketone body production: It increases ketogenesis from 0.35 mM oleate by 90%, from 0.78 mM oleate by 25% and from 1.57 mM butyrate by 37%. alpha-cyano-3-hydroxycinnamate also decreases the activity of tricarboxylic acid cycle and the production of malate and citrate. In hepatocytes from fasted rats, alpha-cyano-3-hydroxycinnamate does not modify ketogenesis from oleate, unless cells are incubated with a mixture of lactate and pyruvate. A lactate and pyruvate mixture decreases ketogenesis from oleate and octanoate and increases citrate and malate production without modifying the uptake of fatty acids. This effect is potentiated by 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase. The results cannot be interpreted only by the effects of malonylCoA on carnitine acyltransferase. They are discussed with respect to the possible involvement of mitochondrial oxaloacetate concentration in the regulation of ketogenesis.

Heat inactivation of leaf phosphoenolpyruvate carboxylase: Protection by aspartate and malate in C4 plants.

PubMed

Rathnam, C K

1978-01-01

The activity of phosphoenolpyruvate (PEP) carboxylase EC 4.1.1.31 in leaf extracts of Eleusine indica L. Gaertn., a C4 plant, exhibited a temperature optimum of 35-37° C with a complete loss of activity at 50° C. However, the enzyme was protected effectively from heat inactivation up to 55° C by L-aspartate. Activation energies (Ea) for the enzyme in the presence of aspartate were 2.5 times lower than that of the control enzyme. Arrhenius plots of PEP carboxylase activity (±aspartate) showed a break in the slope around 17-20° C with a 3-fold increase in the Ea below the break. The discontinuity in the slopes was abolished by treating the enzyme extracts with Triton X-100, suggesting that PEP carboxylase in C4 plants is associated with lipid and may be a membrane bound enzyme. Depending upon the species, the major C4 acid formed during photosynthesis (malate or aspartate) was found to be more protective than the minor C4 acid against the heat inactivation of their PEP carboxylase. Oxaloacetate, the reaction product, was less effective compared to malate or aspartate. Several allosteric inhibitors of PEP carboxylase were found to be moderately to highly effective in protecting the C4 enzyme while its activators showed no significant effect. PEP carboxylase from C3 species was not protected from thermal inactivation by the C4 acids. The physiological significance of these results is discussed in relation to the high temperature tolerance of C4 plants.

Malate as a key carbon source of leaf dark-respired CO2 across different environmental conditions in potato plants.

PubMed

Lehmann, Marco M; Rinne, Katja T; Blessing, Carola; Siegwolf, Rolf T W; Buchmann, Nina; Werner, Roland A

2015-09-01

Dissimilation of carbon sources during plant respiration in support of metabolic processes results in the continuous release of CO2. The carbon isotopic composition of leaf dark-respired CO2 (i.e. δ (13) C R ) shows daily enrichments up to 14.8‰ under different environmental conditions. However, the reasons for this (13)C enrichment in leaf dark-respired CO2 are not fully understood, since daily changes in δ(13)C of putative leaf respiratory carbon sources (δ (13) C RS ) are not yet clear. Thus, we exposed potato plants (Solanum tuberosum) to different temperature and soil moisture treatments. We determined δ (13) C R with an in-tube incubation technique and δ (13) C RS with compound-specific isotope analysis during a daily cycle. The highest δ (13) C RS values were found in the organic acid malate under different environmental conditions, showing less negative values compared to δ (13) C R (up to 5.2‰) and compared to δ (13) C RS of soluble carbohydrates, citrate and starch (up to 8.8‰). Moreover, linear relationships between δ (13) C R and δ (13) C RS among different putative carbon sources were strongest for malate during daytime (r(2)=0.69, P≤0.001) and nighttime (r(2)=0.36, P≤0.001) under all environmental conditions. A multiple linear regression analysis revealed δ (13) C RS of malate as the most important carbon source influencing δ (13) C R . Thus, our results strongly indicate malate as a key carbon source of (13)C enriched dark-respired CO2 in potato plants, probably driven by an anapleurotic flux replenishing intermediates of the Krebs cycle. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Apple fruit acidity is genetically diversified by natural variations in three hierarchical epistatic genes MdSAUR37, MdPP2CH and MdALMTII.

PubMed

Jia, Dongjie; Shen, Fei; Wang, Yi; Wu, Ting; Xu, Xuefeng; Zhang, Xinzhong; Han, Zhenhai

2018-05-11

Many efforts have been made to map quantitative trait loci (QTLs) to facilitate practical marker-assisted selection (MAS) in plants. In the present study, we identified four genome-wide major QTLs responsible for apple fruit acidity by MapQTL and BSA-seq analyses using two independent pedigree-based populations. Candidate genes were screened in major QTL regions, and three functional gene markers, including a non-synonymous A/G single nucleotide polymorphism (SNP) in the coding region of MdPP2CH, a 36-bp insertion in the promoter of MdSAUR37, and a previously reported SNP in MdALMTII, were validated to influence the malate content of apple fruits. In addition, MdPP2CH inactivated three vacuolar H + -ATPases (MdVHA-A3, MdVHA-B2 and MdVHA-D2) and one aluminium-activated malate transporter (MdALMTII) via dephosphorylation and negatively influenced fruit malate accumulation. The dephosphotase activity of MdPP2CH was suppressed by MdSAUR37, which implied a higher hierarchy of genetic interaction. Therefore, the MdSAUR37/MdPP2CH/MdALMTII chain cascaded hierarchical epistatic genetic effects to precisely determine apple fruit malate content. An A/G SNP (-1010) on MdMYB44 promoter region from a major QTL (qtl08.1) was closely associated with fruit malate content. The predicted phenotype values (PPVs) were estimated using the tentative genotype values of the gene markers, and the PPVs were significantly correlated with the observed phenotype values. Our findings provide an insight into plant genome-based selection in apples and will aid in conducting research to understand the physiological fundamentals of quantitative genetics. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Physiological and biochemical performances of menthol-induced aposymbiotic corals.

PubMed

Wang, Jih-Terng; Chen, Yi-Yun; Tew, Kwee Siong; Meng, Pei-Jei; Chen, Chaolun A

2012-01-01

The unique mutualism between corals and their photosynthetic zooxanthellae (Symbiodinium spp.) is the driving force behind functional assemblages of coral reefs. However, the respective roles of hosts and Symbiodinium in this endosymbiotic association, particularly in response to environmental challenges (e.g., high sea surface temperatures), remain unsettled. One of the key obstacles is to produce and maintain aposymbiotic coral hosts for experimental purposes. In this study, a simple and gentle protocol to generate aposymbiotic coral hosts (Isopora palifera and Stylophora pistillata) was developed using repeated incubation in menthol/artificial seawater (ASW) medium under light and in ASW in darkness, which depleted more than 99% of Symbiodinium from the host within 4∼8 days. As indicated by the respiration rate, energy metabolism (by malate dehydrogenase activity), and nitrogen metabolism (by glutamate dehydrogenase activity and profiles of free amino acids), the physiological and biochemical performances of the menthol-induced aposymbiotic corals were comparable to their symbiotic counterparts without nutrient supplementation (e.g., for Stylophora) or with a nutrient supplement containing glycerol, vitamins, and a host mimic of free amino acid mixture (e.g., for Isopora). Differences in biochemical responses to menthol-induced bleaching between Stylophora and Isopora were attributed to the former digesting Symbiodinium rather than expelling the algae live as found in the latter species. Our studies showed that menthol could successfully bleach corals and provided aposymbiotic corals for further exploration of coral-alga symbioses.

Cold acclimation alters DNA methylation patterns and confers tolerance to heat and increases growth rate in Brassica rapa

PubMed Central

Liu, Tongkun; Li, Ying; Duan, Weike; Huang, Feiyi

2017-01-01

Abstract Epigenetic modifications are implicated in plant adaptations to abiotic stresses. Exposure of plants to one stress can induce resistance to other stresses, a process termed cross-adaptation, which is not well understood. In this study, we aimed to unravel the epigenetic basis of elevated heat-tolerance in cold-acclimated Brassica rapa by conducting a genome-wide DNA methylation analysis of leaves from control (CK) and cold-acclimated (CA) plants. We found that both methylation and demethylation occurred during cold acclimation. Two significantly altered pathways, malate dehydrogenase activity and carbon fixation, and 1562 differentially methylated genes, including BramMDH1, BraKAT2, BraSHM4, and Bra4CL2, were identified in CA plants. Genetic validation and treatment of B. rapa with 5-aza-2-deoxycytidine (Aza) suggested that promoter demethylation of four candidate genes increased their transcriptional activities. Physiological analysis suggested that elevated heat-tolerance and high growth rate were closely related to increases in organic acids and photosynthesis, respectively. Functional analyses demonstrated that the candidate gene BramMDH1 (mMDH: mitochondrial malate dehydrogenase) directly enhances organic acids and photosynthesis to increase heat-tolerance and growth rate in Arabidopsis. However, Aza-treated B. rapa, which also has elevated BramMDH1 levels, did not exhibit enhanced heat-tolerance. We therefore suggest that DNA demethylation alone is not sufficient to increase heat-tolerance. This study demonstrates that altered DNA methylation contributes to cross-adaptation. PMID:28158841

A promoter swap strategy between the AtALMT and AtMATE genes increased arabidopsis aluminum resistance and improved carbon use efficiency for aluminum resistance

USDA-ARS?s Scientific Manuscript database

In Arabidopsis, aluminum (Al)-activated AtALMT1-mediated root malate exudation plays a major role in Al tolerance, while Al-activated AtMATE-mediated citrate exudation plays a much smaller role. In this study, we demonstrate that the levels of Al-activated root organic acid exudation are closely co...

Estrogen regulates spatially distinct cardiac mitochondrial subpopulations.

PubMed

Ribeiro Junior, Rogério Faustino; Rodrigues, Paula Lopes; Morra, Elis Aguiar; Ronconi, Karoline Sousa; Do Val Lima, Patrícia Ribeiro; Porto, Marcella Leite; Simões, Maylla Ronacher; Vassallo, Dalton Valentim; Figueiredo, Suely Gomes; Stefanon, Ivanita

2017-07-01

Increased susceptibility to permeability transition pore (mPTP) is a significant concern to decreased cardiac performance in postmenopausal females. The goal of this study was to assess the effects of estrogen deficiency on the two spatially distinct mitochondrial subpopulations from left ventricle: subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM) based on: morphology, membrane potential, oxidative phosphorylation, mPTP and reactive oxygen species production. Female rats (8weeks old) that underwent bilateral ovariectomy were randomly assigned to receive daily treatment with placebo (OVX), estrogen replacement (OVX+E2) and Sham for 60days. The yield for IFM was found higher in the OVX group and lower in the SSM. SSM internal complexity and size were higher in the OVX group, although membrane potential was not different. The maximal rate of mitochondrial respiration, states 3 and 4, using glutamate+malate as substrate, were higher in IFM and SSM from the OVX group. The respiratory control ratio (RCR - state3/state 4), was not different in both SSM and IFM with glutamate+malate. The ADP:O ratio was found lower in IFM and SSM from OVX compared to Sham. When pyruvate was used, state 3 was found unchanged in both IFM and SSM, state 4 was greater in IFM from OVX rats compared to Sham and the ADP/O ratio was decreased. The RCR was unchanged in both subpopulations. The IFM from OVX rats presented a lower Ca 2+ retention capacity compared to Sham, however, the SSM remained unchanged. Hydrogen peroxide formation was found increased in the IFM from OVX animals with glutamate+malate and rotenone+succinate as substrates. The SSM showed increased ROS production only with rotenone+succinate. Western blot analyzes showed decreased levels of PGC-1α and NRF-1 in the OVX group. Estrogen replacement was able to restore most of the alterations induced by ovariectomy. In conclusion, our data shows that estrogen deficiency has distinct effects on the two spatially distinct mitochondrial subpopulations in oxidative phosphorylation, morphology, calcium retention capacity and ROS production. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix

PubMed Central

Brown, Jessica A.; Bulkley, David; Wang, Jimin; Valenstein, Max L.; Yario, Therese A.; Steitz, Thomas A.; Steitz, Joan A.

2014-01-01

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly-abundant nuclear long noncoding RNA that promotes malignancy. A 3′-stem-loop structure is predicted to confer stability by engaging a downstream A-rich tract in a triple helix, similar to the expression and nuclear retention element (ENE) from the KSHV polyadenylated nuclear RNA. The 3.1-Å resolution crystal structure of the human MALAT1 ENE and A-rich tract reveals a bipartite triple helix containing stacks of five and four U•A-U triples separated by a C+•G-C triplet and C-G doublet, extended by two A-minor interactions. In vivo decay assays indicate that this blunt-ended triple helix, with the 3′ nucleotide in a U•A-U triple, inhibits rapid nuclear RNA decay. Interruption of the triple helix by the C-G doublet induces a “helical reset” that explains why triple-helical stacks longer than six do not occur in nature. PMID:24952594

Proteomic analysis of cardiac metabolic enzymes in asphyxiated newborn piglets.

PubMed

Fert-Bober, Justyna; Sawicki, Grzegorz; Lopaschuk, Gary D; Cheung, Po-Yin

2008-11-01

Hypoxia/reoxygenation (H/R) creates an energetic deficiency in the heart, which may contribute to myocardial dysfunction. We hypothesized that H/R-induced impairment of cardioenergetic enzymes occurs in asphyxiated newborn animals. After hypoxia for 2 h (10-15% oxygen), newborn piglets were resuscitated with 100% oxygen for 1 h, followed by 21% oxygen for 3 h. Sham-operated control piglets had no H/R. Hemodynamic parameters in the piglets were continuously measured. At the end of experiment, hearts were isolated for proteomic analysis. In asphyxiated hearts, the level of isocitrate dehydrogenase and malate dehydrogenase was reduced compared to controls. Inverse correlations between the level of myocardial malate dehydrogenase and cardiac function were observed in the control, but not the H/R hearts. We conclude that reoxygenation of asphyxiated newborn piglets reduces the level of myocardial isocitrate dehydrogenase and malate dehydrogenase. While the cause is not clear, it may be related to the impaired tricarboxylic acid cycle pathway and energy production in the heart.

MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples1[OPEN

PubMed Central

Hu, Da-Gang; Sun, Cui-Hui; Ma, Qi-Jun; You, Chun-Xiang; Hao, Yu-Jin

2016-01-01

Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H+-pumping activities of vacuolar H+-ATPase (VHA) and/or vacuolar H+-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H+-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants. PMID:26637549

HvALMT1 from barley is involved in the transport of organic anions

PubMed Central

Gruber, Benjamin D.; Ryan, Peter R.; Richardson, Alan E.; Tyerman, Stephen D.; Ramesh, Sunita; Hebb, Diane M.; Howitt, Susan M.; Delhaize, Emmanuel

2010-01-01

Members of the ALMT gene family contribute to the Al3+ resistance of several plant species by facilitating malate efflux from root cells. The first member of this family to be cloned and characterized, TaALMT1, is responsible for most of the natural variation of Al3+ resistance in wheat. The current study describes the isolation and characterization of HvALMT1, the barley gene with the greatest sequence similarity to TaALMT1. HvALMT1 is located on chromosome 2H which has not been associated with Al3+ resistance in barley. The relatively low levels of HvALMT1 expression detected in root and shoot tissues were independent of external aluminium or phosphorus supply. Transgenic barley plants transformed with the HvALMT1 promoter fused to the green fluorescent protein (GFP) indicated that expression of HvALMT1 was relatively high in stomatal guard cells and in root tissues containing expanding cells. GFP fused to the C-terminus of the full HvALMT1 protein localized to the plasma membrane and motile vesicles within the cytoplasm. HvALMT1 conferred both inward and outward currents when expressed in Xenopus laevis oocytes that were bathed in a range of anions including malate. Both malate uptake and efflux were confirmed in oocyte assays using [14C]malate as a radiotracer. It is suggested that HvALMT1 functions as an anion channel to facilitate organic anion transport in stomatal function and expanding cells. PMID:20176888

HvALMT1 from barley is involved in the transport of organic anions.

PubMed

Gruber, Benjamin D; Ryan, Peter R; Richardson, Alan E; Tyerman, Stephen D; Ramesh, Sunita; Hebb, Diane M; Howitt, Susan M; Delhaize, Emmanuel

2010-03-01

Members of the ALMT gene family contribute to the Al(3+) resistance of several plant species by facilitating malate efflux from root cells. The first member of this family to be cloned and characterized, TaALMT1, is responsible for most of the natural variation of Al(3+) resistance in wheat. The current study describes the isolation and characterization of HvALMT1, the barley gene with the greatest sequence similarity to TaALMT1. HvALMT1 is located on chromosome 2H which has not been associated with Al(3+) resistance in barley. The relatively low levels of HvALMT1 expression detected in root and shoot tissues were independent of external aluminium or phosphorus supply. Transgenic barley plants transformed with the HvALMT1 promoter fused to the green fluorescent protein (GFP) indicated that expression of HvALMT1 was relatively high in stomatal guard cells and in root tissues containing expanding cells. GFP fused to the C-terminus of the full HvALMT1 protein localized to the plasma membrane and motile vesicles within the cytoplasm. HvALMT1 conferred both inward and outward currents when expressed in Xenopus laevis oocytes that were bathed in a range of anions including malate. Both malate uptake and efflux were confirmed in oocyte assays using [(14)C]malate as a radiotracer. It is suggested that HvALMT1 functions as an anion channel to facilitate organic anion transport in stomatal function and expanding cells.

Effect of pH on optimization of photofermentative hydrogen production by co-culture of Rhodobacter sphaeroides-NMBL-02 and Bacillus firmus-NMBL-03.

PubMed

Pandey, A; Dolly, S; Semwal, D; Pandey, A

2017-07-31

Rhodobacter sphaeroides NMBL-02, photosynthetic purple non sulfur (PNS) bacteria and associated Bacillus firmus NMBL-03 were isolated from water sample collected from 15-20 inches beneath the surface of ponds from Northern region of India in modified Sistrom's media (120 ml) containing 3 g/L malate and 1.2 g/L ammonium sulfate. The isolation was done in air tight serum bottles (120 ml) under tungsten bulb (1.8 kLux light intensity) at 30 oC ± 2 oC. The PNS and heterotrophic bacteria associated with the culture was purified by clonal selection method and characterized by 16S rDNA sequencing. The PNS isolate was identified as Rhodobacter sphaeroides NMBL-02 (ID: 1467407, Accession BANKIT: JN256030) and associated heterotroph as Bacillus firmus NMBL-03 (Gene Bank Accession no.: JN 256029). The effect of initial medium pH on optimization of hydrogen production was investigated in batch process. The maximum hydrogen potential and hydrogen production rate was 2310 ± 55 ml/L and 4.75 ml/L culture/h respectively using glutamate (1.7 mmol/L) as nitrogen source and malate (22.38 mmol/L) as carbon source with 76.39% malate conversion efficiency at initial medium pH 5.0. This co-culture has the ability to produce significant amount of hydrogen in the pH range of 5.0 to 10.0 with 76.39% to 35.71% malate conversion respectively.

MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples.

PubMed

Hu, Da-Gang; Sun, Cui-Hui; Ma, Qi-Jun; You, Chun-Xiang; Cheng, Lailiang; Hao, Yu-Jin

2016-03-01

Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H(+)-pumping activities of vacuolar H(+)-ATPase (VHA) and/or vacuolar H(+)-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H(+)-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants. © 2016 American Society of Plant Biologists. All Rights Reserved.

Antisense Suppression of the Small Chloroplast Protein CP12 in Tobacco Alters Carbon Partitioning and Severely Restricts Growth1[W

PubMed Central

Howard, Thomas P.; Fryer, Michael J.; Singh, Prashant; Metodiev, Metodi; Lytovchenko, Anna; Obata, Toshihiro; Fernie, Alisdair R.; Kruger, Nicholas J.; Quick, W. Paul; Lloyd, Julie C.; Raines, Christine A.

2011-01-01

The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. The importance of CP12 in vivo in higher plants, however, has not been investigated. Here, antisense suppression of CP12 in tobacco (Nicotiana tabacum) was observed to impact on NAD-induced PRK and GAPDH complex formation but had little effect on enzyme activity. Additionally, only minor changes in photosynthetic carbon fixation were observed. Despite this, antisense plants displayed changes in growth rates and morphology, including dwarfism and reduced apical dominance. The hypothesis that CP12 is essential to separate oxidative pentose phosphate pathway activity from Calvin-Benson cycle activity, as proposed in cyanobacteria, was tested. No evidence was found to support this role in tobacco. Evidence was seen, however, for a restriction to malate valve capacity, with decreases in NADP-malate dehydrogenase activity (but not protein levels) and pyridine nucleotide content. Antisense repression of CP12 also led to significant changes in carbon partitioning, with increased carbon allocation to the cell wall and the organic acids malate and fumarate and decreased allocation to starch and soluble carbohydrates. Severe decreases were also seen in 2-oxoglutarate content, a key indicator of cellular carbon sufficiency. The data presented here indicate that in tobacco, CP12 has a role in redox-mediated regulation of carbon partitioning from the chloroplast and provides strong in vivo evidence that CP12 is required for normal growth and development in plants. PMID:21865489

Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea1[W][OA

PubMed Central

Shane, Michael W.; Fedosejevs, Eric T.; Plaxton, William C.

2013-01-01

Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al3+, Fe3+, and Ca2+ phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme’s activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced Km [PEP] coupled with elevated I50 [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots. PMID:23407057

Noncitrus alkaline fruit: a dietary alternative for the treatment of hypocitraturic stone formers.

PubMed

Baia, Leandro da Cunha; Baxmann, Alessandra Calábria; Moreira, Silvia Regina; Holmes, Ross Philip; Heilberg, Ita Pfeferman

2012-09-01

Fruits and vegetables are natural suppliers of potassium, bicarbonate, or bicarbonate precursors such as citrate, malate and others-hence, possessing potential effects on citraturia. We aimed to compare the acute effects of a noncitrus (melon) fruit vs citric ones (orange and lime) on citraturia and other lithogenic parameters. Two-hour urine samples were collected from 30 hypocitraturic stone-forming patients after an overnight fast and 2, 4, and 6 hours after the consumption of 385 mL (13 oz) of either freshly squeezed orange juice (n=10), freshly blended melon juice (n=10), or freshly squeezed lime juice (n=10). Urinary citrate, potassium, pH, and other lithogenic parameters were determined and net gastrointestinal alkali absorption (NGIA) was calculated. Potential renal acid load (PRAL) and pH from juices were determined. Significant and comparable increases of mean urinary citrate were observed in all groups, whereas mean urinary potassium, pH, and NGIA were significantly increased only after consumption of melon and orange juices. The pH of melon juice was higher and the PRAL value was more negative compared with orange juice, indicating a higher alkalinity. These findings suggested that melon, a noncitrus source of potassium, citrate, and malate, yielded an increase in urinary citrate excretion equivalent to that provided by orange, and hence represents another dietary alternative for the treatment of hypocitraturic stone-formers. Despite its low potassium content, lime also produced comparable increases in citraturia possibly because of its high citric acid content.

Anaerobic Carbon Metabolism by the Tricarboxylic Acid Cycle 1

PubMed Central

Vanlerberghe, Greg C.; Horsey, Anne K.; Weger, Harold G.; Turpin, David H.

1989-01-01

Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH4+ in the dark under anaerobic conditions. Addition of NH4+ to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO2 efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenolpyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H14CO3− to anaerobic cells assimilating NH4+ results in the incorporation of radiolabel into the α-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH4+ addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply α-ketoglutarate for glutamate production. During dark aerobic NH4+ assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH4+ assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH4+ assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity. PMID:16667215

Calcium signaling in brain mitochondria: interplay of malate aspartate NADH shuttle and calcium uniporter/mitochondrial dehydrogenase pathways.

PubMed

Contreras, Laura; Satrústegui, Jorgina

2009-03-13

Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.

Direct effects of Vaccinium myrtillus L. fruit extracts on rat heart mitochondrial functions.

PubMed

Trumbeckaitė, S; Burdulis, D; Raudonė, L; Liobikas, J; Toleikis, A; Janulis, V

2013-04-01

In this study, the direct influence of bilberry (Vaccinium myrtillus) fruit extracts (aqueous and ethanolic) rich in anthocyanins on the oxidative phosphorylation of isolated rat heart mitochondria was investigated in vitro. Higher concentrations of bilberry extracts concentration-dependently inhibited mitochondrial state 3 respiration (by 23%-61%) with pyruvate plus malate, mildly (by 1.2- to 1.3-fold) uncoupled the oxidative phosphorylation, and increased (by 30%-87%) the state 4 respiration rate in the presence of exogenous cytochrome c. Succinate oxidation was less affected. Pure anthocyanins, the main components of used extracts, malvidin-3-glucoside, malvidin-3-galactoside, and cyanidin-3-galactoside, had no effect on oxidation of pyruvate plus malate. A statistically significant decrease in H2 O2 production by mitochondria was found in the presence of bilberry fruit extracts. Our findings show that bilberry fruit anthocyanin-rich extracts possess direct effects on rat heart mitochondrial function in vitro. These findings give the first insights into the mechanism(s) of their action on cellular energy metabolism. Copyright © 2012 John Wiley & Sons, Ltd.

Malate decarboxylases: evolution and roles of NAD(P)-ME isoforms in species performing C(4) and C(3) photosynthesis.

PubMed

Maier, Alexandra; Zell, Martina B; Maurino, Veronica G

2011-05-01

In the C(4) pathway of photosynthesis two types of malate decarboxylases release CO(2) in bundle sheath cells, NADP- and NAD-dependent malic enzyme (NADP-ME and NAD-ME), located in the chloroplasts and the mitochondria of these cells, respectively. The C(4) decarboxylases involved in C(4) photosynthesis did not evolve de novo; they were recruited from existing housekeeping isoforms. NADP-ME housekeeping isoforms would function in the control of malate levels during hypoxia, pathogen defence responses, and microspore separation, while NAD-ME participates in the respiration of malate in the tricarboxylic acid cycle. Recently, the existence of three enzymatic NAD-ME entities in Arabidopsis, occurring by alternative association of two subunits, was described as a novel mechanism to regulate NAD-ME activity under changing metabolic environments. The C(4) NADP-ME is thought to have evolved from a C(3) chloroplastic ancestor, which in turn would have evolved from an ancient cytosolic enzyme. In this way, the C(4) NADP-ME would have emerged through gene duplication, acquisition of a new promoter, and neo-functionalization. In contrast, there would exist a unique NAD-ME in C(4) plants, which would have been adapted to perform a dual function through changes in the kinetic and regulatory properties of the C(3) ancestors. In addition to this, for the evolution of C(4) NAD-ME, insertion of promoters or enhancers into the single-copy genes of the C(3) ancestors would have changed the expression without gene duplication.

Development of an Amperometric Biosensor Platform for the Combined Determination of L-Malic, Fumaric, and L-Aspartic Acid.

PubMed

Röhlen, Désirée L; Pilas, Johanna; Schöning, Michael J; Selmer, Thorsten

2017-10-01

Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD + ) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM -1 (L-malate biosensor) and 0.4 μA mM -1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0-10.0 mM with a sensitivity of 0.09 μA mM -1 . The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.

Accumulation of nonesterified fatty acids causes the sustained energetic deficit in kidney proximal tubules after hypoxia-reoxygenation.

PubMed

Feldkamp, Thorsten; Kribben, Andreas; Roeser, Nancy F; Senter, Ruth A; Weinberg, Joel M

2006-02-01

Kidney proximal tubules exhibit decreased ATP and reduced, but not absent, mitochondrial membrane potential (Deltapsi(m)) during reoxygenation after severe hypoxia. This energetic deficit, which plays a pivotal role in overall cellular recovery, cannot be explained by loss of mitochondrial membrane integrity, decreased electron transport, or compromised F1F0-ATPase and adenine nucleotide translocase activities. Addition of oleate to permeabilized tubules produced concentration-dependent decreases of Deltapsi(m) measured by safranin O uptake (threshold for oleate = 0.25 microM, 1.6 nmol/mg protein; maximal effect = 4 microM, 26 nmol/mg) that were reversed by delipidated BSA (dBSA). Cell nonesterified fatty acid (NEFA) levels increased from <1 to 17.4 nmol/mg protein during 60- min hypoxia and remained elevated at 7.6 nmol/mg after 60 min reoxygenation, at which time ATP had recovered to only 10% of control values. Safranin O uptake in reoxygenated tubules, which was decreased 85% after 60-min hypoxia, was normalized by dBSA, which improved ATP synthesis as well. dBSA also almost completely normalized Deltapsi(m) when the duration of hypoxia was increased to 120 min. In intact tubules, the protective substrate combination of alpha-ketoglutarate + malate (alpha-KG/MAL) increased ATP three- to fourfold, limited NEFA accumulation during hypoxia by 50%, and lowered NEFA during reoxygenation. Notably, dBSA also improved ATP recovery when added to intact tubules during reoxygenation and was additive to the effect of alpha-KG/MAL. We conclude that NEFA overload is the primary cause of energetic failure of reoxygenated proximal tubules and lowering NEFA substantially contributes to the benefit from supplementation with alpha-KG/MAL.

Modeling, molecular docking, probing catalytic binding mode of acetyl-CoA malate synthase G in Brucella melitensis 16M.

PubMed

Adi, Pradeepkiran Jangampalli; Yellapu, Nanda Kumar; Matcha, Bhaskar

2016-12-01

There are enormous evidences and previous reports standpoint that the enzyme of glyoxylate pathway malate synthase G (MSG) is a potential virulence factor in several pathogenic organisms, including Brucella melitensis 16M. Where the lack of crystal structures for best candidate proteins like MSG of B. melitensis 16M creates big lacuna to understand the molecular pathogenesis of brucellosis. In the present study, we have constructed a 3-D structure of MSG of Brucella melitensis 16M in MODELLER with the help of crystal structure of Mycobacterium tuberculosis malate synthase (PDB ID: 2GQ3) as template. The stereo chemical quality of the restrained model was evaluated by SAVES server; remarkably we identified the catalytic functional core domain located at 4 th cleft with conserved catalytic amino acids, start at ILE 59 to VAL 586 manifest the function of the protein. Furthermore, virtual screening and docking results reveals that best leadmolecules binds at the core domain pocket of MSG catalytic residues and these ligand leads could be the best prospective inhibitors to treat brucellosis.

[Different NaCl-dependence of the circadian CO2-gas-exchange of some halophil growing coastal plants].

PubMed

Treichel, Siegfried; Bauer, Peter

1974-03-01

CO 2 -exchange, diurnal changes in malate- and ion concentrations of the halophytes Carpobrotus edulis, Crithmum maritimum, Mesembryanthemum nodiflorum, Salicornia fruticosa, Suaeda maritima, and Trifolium fragiferum were investigated after culture at different NaCl concentrations. In Carp. edulis and Mes. nodiflorum the diurnal rhythm of CO 2 -exchange is in accordance with that of crassulacean acid metabolism (CAM), in Sal. fruticosa, Crithm. maritimum, Suaeda maritima, and Trif. fragiferum with that of Benson-Calvin metabolism (C 3 ). Malate concentration and CO 2 uptake in the sap latter group are not influenced. On the other hand, Carp. edulis and Mes. nodiflorum show an accumulation of malate during the night, which can be interpreted as a further indication of CAM.The two species most resistant to NaCl, Carp. edulis and Sal. fruticosa, greatly differ very much in their NaCl content. NaCl concentration in Salicornia is four times higher than in Carpobrotus.The different metabolic properties studied might be of ecological importance for the plants in their natural habitats. The effect of NaCl on metabolic processes is discussed.

The Epiphytic Fern Elaphoglossum luridum (Fée) Christ. (Dryopteridaceae) from Central and South America: Morphological and Physiological Responses to Water Stress

PubMed Central

Minardi, Bruno Degaspari; Voytena, Ana Paula Lorenzen; Randi, Áurea Maria

2014-01-01

Elaphoglossum luridum (Fée) Christ. (Dryopteridaceae) is an epiphytic fern of the Atlantic Forest (Brazil). Anatomical and physiological studies were conducted to understand how this plant responds to water stress. The E. luridum frond is coriaceus and succulent, presenting trichomes, relatively thick cuticle, and sinuous cell walls in both abaxial and adaxial epidermis. Three treatments were analyzed: control, water deficit, and abscisic acid (ABA). Physiological studies were conducted through analysis of relative water content (RWC), photosynthetic pigments, chlorophyll a fluorescence, and malate content. No changes in RWC were observed among treatments; however, significant decreases in chlorophyll a content and photosynthetic parameters, including optimal irradiance (I opt) and maximum electron transport rate (ETRmax), were determined by rapid light curves (RLC). No evidence of crassulacean acid metabolism (CAM) pathway was observed in E. luridum in response to either water deficit or exogenous application of ABA. On the other hand, malate content decreased in the E. luridum frond after ABA treatment, seeming to downregulate malate metabolism at night, possibly through tricarboxylic acid (TCA) cycle regulation. PMID:25386618

Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger.

PubMed

Kirimura, Kohtaro; Kobayashi, Keiichi; Ueda, Yuka; Hattori, Takasumi

2016-09-01

The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.

[A case of respiratory dyskinesia due to clebopride malate].

PubMed

Kawasaki, H; Yamamoto, M; Okayasu, H; Wakayama, Y

1991-08-01

Clebopride malate is therapeutically used for the treatment of peptic ulcer. This drug has potent antidopaminergic activity that causes acute dystonic reaction, parkinsonism and tardive dyskinesia as adverse effects. Here, we have reported an 86-year-old man who developed abnormal involuntary movement of respiratory muscles and lower limb muscles after this drug had been given for four months. This involuntary movement appeared spontaneously at resting state and disappeared during sleep. Surface EMG demonstrated a synchronous grouping discharge in m. orbicularis oris, m. sternocleidomastoideus and m. interstales which synchronized with diaphragmatic movement on cinefluorography. Involuntary movement of the lower limbs was synchronous bilaterally and had little relationship with diaphragmatic movement. This involuntary movement was irregular not only in rhythm but also in duration. According to this irregular nature, we diagnosed this involuntary movement as respiratory dyskinesia with limb dyskinesia that belongs to tardive dyskinesia. After cessation of clebopride malate limb dyskinesia disappeared rapidly and respiratory dyskinesia markedly decreased. We emphasize that respiratory dyskinesia should be differentiated from psychogenic hyperventilation as easily misdiagnosed on initial examination.

Expression of the long non-coding RNA H19 and MALAT-1 in growth hormone-secreting pituitary adenomas and its relationship to tumor behavior.

PubMed

Lu, Tianyu; Yu, Chen; Ni, Hongbin; Liang, Weibang; Yan, Huiying; Jin, Wei

2018-06-01

Aggressive growth hormone-secreting pituitary adenomas (GHPAs) represent an important clinical problem in patients with acromegaly. Surgical therapy, although often the mainstay of treatment for GHPAs, is less effective in aggressive GHPAs due to their invasive and destructive growth patterns, and their proclivity for infrasellar invasion. LncRNAs are important players in cancer development and emerging in various fundamental biological processes. In the present study, qRT-PCR was performed to examine the expression of lncRNA H19 and MALAT-1 in invasive and non-invasive GHPAs. Our results revealed that the expression of lncRNA H19 was remarkably higher in invasive GHPAs, however, there was no significant differences between the expression of lncRNA MALAT-1 in invasive GHPAs and non-invasive GHPAs, suggesting that lncRNA H19 may play an important role in GHPA invasion. LncRNA H19 might be a target for the study of GHPAs invasion, and a potential index for the diagnosis or prognosis of GHPAs. Copyright © 2018. Published by Elsevier Ltd.

Isolation and Oxidative Properties of Intact Mitochondria Isolated from Spinach Leaves 1

PubMed Central

Douce, Roland; Moore, Antony L.; Neuburger, Michel

1977-01-01

A procedure was described for preparing intact mitochondria from spinach (Spinacia oleracea L.) leaves. These mitochondria oxidized succinate, malate, pyruvate, α-ketoglutarate, and NADH with good respiratory control and ADP/O ratios comparable to those observed with mitochondria from other plant tissues. Glycine was oxidized by the preparations. This oxidation linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport and phosphorylation inhibitors. Cyanide completely inhibited the oxidation of NADH. The oxidation of succinate, malate, and glycine was only partially inhibited. Images PMID:16660151

Effect of silicon on wheat seedlings (Triticum turgidum L.) grown in hydroponics and exposed to 0 to 30 µM Cu.

PubMed

Keller, C; Rizwan, M; Davidian, J-C; Pokrovsky, O S; Bovet, N; Chaurand, P; Meunier, J-D

2015-04-01

Aqueous Si limits Cu uptake by a Si-accumulating plant via physicochemical mechanisms occurring at the root level. Sufficient Si supply may alleviate Cu toxicity in Cu-contaminated soils. Little information is available on the role of silicon (Si) in copper (Cu) tolerance while Cu toxicity is widespread in crops grown on Cu-contaminated soils. A hydroponic study was set up to investigate the influence of Si on Cu tolerance in durum wheat (Triticum turgidum L.) grown in 0, 0.7, 7.0 and 30 µM Cu without and with 1.0 mM Si, and to identify the mechanisms involved in mitigation of Cu toxicity. Si supply alleviated Cu toxicity in durum wheat at 30 µM Cu, while Cu significantly increased Si concentration in roots. Root length, photosynthetic pigments concentrations, macroelements, and organic anions (malate, acetate and aconitate) in roots, were also increased. Desorption experiments, XPS analysis of the outer thin root surface (≤100 Å) and µXRF analyses showed that Si increased adsorption of Cu at the root surface as well as Cu accumulation in the epidermis while Cu was localised in the central cylinder when Si was not applied. Copper was not detected in phytoliths. This study provides evidences for Si-mediated alleviation of Cu toxicity in durum wheat. It also shows that Si supplementation to plants exposed to increasing levels of Cu in solution induces non-simultaneous changes in physiological parameters. We propose a three-step mechanism occurring mainly at the root level and limiting Cu uptake and translocation to shoots: (i) increased Cu adsorption onto the outer thin layer root surface and immobilisation in the vicinity of root epidermis, (ii) increased Cu complexation by both inorganic and organic anions such as aconitate and, (iii) limitation of translocation through an enhanced thickening of a Si-loaded endodermis.

WIF1 re-expression in glioblastoma inhibits migration through attenuation of non-canonical WNT signaling by downregulating the lncRNA MALAT1.

PubMed

Vassallo, I; Zinn, P; Lai, M; Rajakannu, P; Hamou, M-F; Hegi, M E

2016-01-07

Glioblastoma is the most aggressive primary brain tumor in adults and due to the invasive nature cannot be completely removed. The WNT inhibitory factor 1 (WIF1), a secreted inhibitor of WNTs, is systematically downregulated in glioblastoma and acts as strong tumor suppressor. The aim of this study was the dissection of WIF1-associated tumor-suppressing effects mediated by canonical and non-canonical WNT signaling. We found that WIF1 besides inhibiting the canonical WNT pathway selectively downregulates the WNT/calcium pathway associated with significant reduction of p38-MAPK (p38-mitogen-activated protein kinase) phosphorylation. Knockdown of WNT5A, the only WNT ligand overexpressed in glioblastoma, phenocopied this inhibitory effect. WIF1 expression inhibited cell migration in vitro and in an orthotopic brain tumor model, in accordance with the known regulatory function of the WNT/Ca(2+) pathway on migration and invasion. In search of a mediator for this function differential gene expression profiles of WIF1-expressing cells were performed. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA and key positive regulator of invasion, emerged as the top downregulated gene. Indeed, knockdown of MALAT1 reduced migration in glioblastoma cells, without effect on proliferation. Hence, loss of WIF1 enhances the migratory potential of glioblastoma through WNT5A that activates the WNT/Ca(2+) pathway and MALAT1. These data suggest the involvement of canonical and non-canonical WNT pathways in glioblastoma promoting key features associated with this deadly disease, proliferation on one hand and invasion on the other. Successful targeting will require a dual strategy affecting both canonical and non-canonical WNT pathways.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

PubMed

Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

2015-08-21

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

PubMed Central

Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

2015-01-01

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

Anaerobic carbon metabolism by the tricarboxylic acid cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vanlerberghe, G.C.; Horsey, A.K.; Weger, H.G.

Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH{sub 4}{sup +} in the dark under anaerobic conditions. Addition of NH{sub 4}{sup +} to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO{sub 2} efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenspyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H{sup 14}CO{sub 3}{sup {minus}} to anaerobic cells assimilating NH{sub 4}{sup +} results in the incorporation of radiolabel into the {alpha}-carboxyl carbon of glutamic acid. Incorporationmore » of radiolabel into glutamic acid is not simply a short-term phenomenon following NH{sub 4}{sup +} addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply {alpha}ketoglutarate for glutamate production. During dark aerobic NH{sub 4}{sup +} assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH{sub 4}{sup +} assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH{sub 4}{sup +} assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity.« less

Anaerobic Carbon Metabolism by the Tricarboxylic Acid Cycle : Evidence for Partial Oxidative and Reductive Pathways during Dark Ammonium Assimilation.

PubMed

Vanlerberghe, G C; Horsey, A K; Weger, H G; Turpin, D H

1989-12-01

Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH(4) (+) in the dark under anaerobic conditions. Addition of NH(4) (+) to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO(2) efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenolpyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H(14)CO(3) (-) to anaerobic cells assimilating NH(4) (+) results in the incorporation of radiolabel into the alpha-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH(4) (+) addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply alpha-ketoglutarate for glutamate production. During dark aerobic NH(4) (+) assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH(4) (+) assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH(4) (+) assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity.

Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium.

PubMed Central

Stevens, T O; Linkfield, T G; Tiedje, J M

1988-01-01

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium. PMID:3223760

Eighteen-deoxyaldosterone and other less polar forms of 18-hydroxycorticosterone as aldosterone precursors in rat adrenals.

PubMed

Cozza, E N; Burton, G; Ceballos, N R; Lantos, C P; Harnik, M; Scott, A I

1985-05-01

Samples containing as precursors either 18-hydroxycorticosterone (18-OH-B) in its M form, or this converted to less polar forms at pH 2 (ACM), or M or ACM enclosed in liposomes from adrenal lipids were incubated at pH 7.4, 4.8 or 3.3 in the presence or absence of quartered rat adrenals for 1 and 2 h. Optimal (10%) yields of aldosterone were obtained when (a) ACM was incubated at pH 4.8 and (b) M enclosed in liposomes was suspended in buffer and shaken without enzyme at pH 3.3. When conditions (a) were supplemented with malate and NADP, 16% of ACM was converted to aldosterone. ACM contained 80% of a fraction which, according to 13C NMR spectroscopy, was identical to 18-deoxyaldosterone (18-DAL). Experiments in which radioactivity from corticosterone (B) or M was trapped by radioinert M or 18-DAL disclosed a pathway comprising sequentially B, 18-OH-B, 18-DAL and aldosterone, and the combined evidence of this work, an enzymatic hydroxylation of 18-DAL to aldosterone.

Evaluation of mitochondrial activity by two-photon absorption with near-field multioptical fiber probes

NASA Astrophysics Data System (ADS)

Kanazashi, Yasuaki; Takara, Naoshi; Iwami, Kentaro; Ohta, Yoshihiro; Umeda, Norihiro

2018-04-01

pH measurements enable the direct monitoring and evaluation of mitochondrial activity. We constructed a scanning near-field optical microscopy system with multioptical fiber probes using the two-photon absorption of a pH-sensitive fluorescent dye, SNARF-4F, to measure the activity difference of mitochondrial aggregates. pH can be monitored through the fluorescence intensity ratio (FIR) of SNARF-4F. We derived a calibration curve of the FIR as a function of pH. The FIR dynamic responses were measured by adding hydrochloric acid to the buffer solution. Using the developed system, we simultaneously measured the pH changes at two different locations in the SNARF-4F solution. Mitochondrial samples were prepared using optical tweezers to control the number and position of mitochondria. Mitochondrial pH changes (ΔpH) between 0.05 and 0.57 were observed after adding a nutritional supplement (malate and glutamate). In addition, in the comparative experiment on the activities of two mitochondrial populations, the obtained result suggested that the activity differs depending on the difference in the number of mitochondria.

Plant, cell, and molecular mechanisms of abscisic-acid regulation of stomatal apertures. In vivo phosphorylation of phosphoenolpyruvate carboxylase in guard cells of Vicia faba L. is enhanced by fusicoccin and suppressed by abscisic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Z.; Aghoram, K.; Outlaw, W.H. Jr.

Plants regulate water loss and CO{sub 2} gain by modulating the aperture sizes of stomata that penetrate the epidermis. Aperture size itself is increased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma. Guard-cell phosphoenolpyruvate carboxylase, which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Regulation of this cytosolic enzyme by effectors is well documented, but additional regulation by posttranslational modification is predicted by the alteration of PEPC kinetics during stomatal opening. In this study, the authors have investigated whether this alterationmore » is associated with the phosphorylation status of this enzyme. Using sonicated epidermal peels (isolated guard cells) pre-loaded with {sub 32}PO{sub 4}, the authors induced stomatal opening and guard-cell malate accumulation by incubation with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 {micro}M abscisic acid (ABA). The phosphorylation status of PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. PEPC was phosphorylated when stomata were stimulated to open, and phosphorylation was lessened by incubation with ABA.« less

13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Amit; Ando, David; Gin, Jennifer

Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. Thesemore » genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.« less

13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

DOE PAGES

Ghosh, Amit; Ando, David; Gin, Jennifer; ...

2016-10-05

Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. Thesemore » genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.« less

Some enzymes of carbohydrate metabolism in Mesocestoides corti and Heterakis spumosa.

PubMed

Dubinský, P; Ruscinová, B; Hetmanski, S L; Arme, C; Turceková, L; Rybos, M

1991-09-01

The activities of selected enzymes of carbohydrate metabolism were measured in tetrathyridia of Mesocestoides corti and in adult females and males of Heterakis spumosa. When the species were compared, only lactate dehydrogenase and phosphoenolpyruvate carboxykinase activities were considerably higher in M. corti. Activities of other enzymes were higher in H. spumosa, with malate dehydrogenase activity being considerably so. In H. spumosa, enzyme activity was higher, and succinate dehydrogenase markedly so in males, when compared with females. Tetrathyridia aged 170 and 210 days show relatively stable malate and lactate dehydrogenase activities, and mice of ICR and BALB/c strains are suitable for the maintenance of tetrathyridia.

The role of dietary long chain fatty acids in mitochondrial structure and function. Effects on rat cardiac mitochondrial respiration.

PubMed

Clandinin, M T

1978-02-01

To evaluate the effect of dietary rapeseed oils on cardiac mitochondrial function and metabolic conservation of energy, male weanling rats derived from the Sprague-Dawley strain were fed three rations containing either 15% (w/w) soybean oil, low erucic acid rapeseed oil or a high erucic acid rapeseed oil. Cardiac mitochondria were isolated for measurement of mitochondrial respiratory functions. Pyruvate and malate plus malonate or succinate plus amytal, or alpha-ketoglutarate and malate plus malonate were utilized as substrates for oxidative phosphorylation. Net rates of state 3 oxygen uptake and therefore ATP synthesis were found to decline with chronic feeding of the 15% (w/w) oil containing diets. Significantly reduced ADP/O ratios were observed for groups fed high erucic acid rapeseed oil containing diets for 11 days. Decreased ADP/O ratios were also observed for groups fed high or low erucic acid rapeseed oils for 112 days. When pyruvate and malate plus malonate were utilized as substrates, reduced rates of ATP synthesis were observed after chronic feeding of high erucic acid rapeseed oil diets. Only prolonged feeding of low erucic acid rapeseed oils resulted in significant alterations in the efficiency of oxidative phosphorylation.

Oxaloacetate Transport into Plant Mitochondria1

PubMed Central

Hanning, Iris; Baumgarten, Katharina; Schott, Karin; Heldt, Hans W.

1999-01-01

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis. PMID:10069840

Sunitinib: from charge-density studies to interaction with proteins.

PubMed

Malińska, Maura; Jarzembska, Katarzyna N; Goral, Anna M; Kutner, Andrzej; Woźniak, Krzysztof; Dominiak, Paulina M

2014-05-01

Protein kinases are targets for the treatment of a number of diseases. Sunitinib malate is a type I inhibitor of tyrosine kinases and was approved as a drug in 2006. This contribution constitutes the first comprehensive analysis of the crystal structures of sunitinib malate and of complexes of sunitinib with a series of protein kinases. The high-resolution single-crystal X-ray measurement and aspherical atom databank approach served as a basis for reconstruction of the charge-density distribution of sunitinib and its protein complexes. Hirshfeld surface and topological analyses revealed a similar interaction pattern in the sunitinib malate crystal structure to that in the protein binding pockets. Sunitinib forms nine preserved bond paths corresponding to hydrogen bonds and also to the C-H···O and C-H···π contacts common to the VEGRF2, CDK2, G2, KIT and IT kinases. In general, sunitinib interacts with the studied proteins with a similar electrostatic interaction energy and can adjust its conformation to fit the binding pocket in such a way as to enhance the electrostatic interactions, e.g. hydrogen bonds in ligand-kinase complexes. Such behaviour may be responsible for the broad spectrum of action of sunitinib as a kinase inhibitor.

Mechanisms of respiration intensification of rat pancreatic acini upon carbachol-induced Ca(2+) release.

PubMed

Manko, B O; Manko, V V

2013-08-01

Acetylcholine as one of the main secretagogues modulates mitochondrial functions in acinar pancreacytes, presumably due to increase in ATP hydrolysis or Ca(2+) transport into mitochondria. The aim of this work was to investigate the mechanisms of carbachol (CCh) action on respiration and oxidative phosphorylation of isolated pancreatic acini. Respiration of intact or permeabilized rat pancreatic acini was studied at 37 °C using a Clark oxygen electrode. Respiration rate of isolated acini in rest was 0.27 ± 0.01 nmol O2 s(-1) 10(-6) cells. Addition of 10 μM CCh into respiration chamber evoked biphasic stimulation of respiration. Rapid increase of respiration by 20.1% lasted for approx. 1 min, followed by decrease to level by 11.5% higher than control. Addition of 1 μm CCh caused monophasic increase by 11.5%. Preincubation (5 min) with 1 or 10 μm CCh elevated respiration rate by 12.5 or 11.2% respectively. FCCP prevented the effect of CCh. Preincubation with 1 (but not 10) μm CCh increased FCCP-uncoupled respiration rate. Thapsigargin slightly elevated respiration, but ryanodine did not. Application of 2-aminoethoxydiphenyl borate or ruthenium red prevented the effects of CCh on respiration, while oligomycin abolished them. Preincubation with 1 μm CCh prior to cell permeabilization increased respiration rate at pyruvate+malate oxidation, but not at succinate oxidation. In contrast, preincubation with 10 μm CCh decreased pyruvate+malate oxidation. Medium CCh dose (1 μm) intensifies respiration and oxidative phosphorylation of acinar pancreacytes by feedforward mechanism via Ca(2+) transport into mitochondria and activation of Ca(2+) /ADP-sensitive mitochondrial dehydrogenases. Prolonged action of high CCh dose (10 μm) might impair mitochondrial functions. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Long-lived mitochondrial (Mit) mutants of Caenorhabditis elegans utilize a novel metabolism.

PubMed

Butler, Jeffrey A; Ventura, Natascia; Johnson, Thomas E; Rea, Shane L

2010-12-01

The Caenorhabditis elegans mitochondrial (Mit) mutants have disrupted mitochondrial electron transport chain (ETC) functionality, yet, surprisingly, they are long lived. We have previously proposed that Mit mutants supplement their energy needs by exploiting alternate energy production pathways normally used by wild-type animals only when exposed to hypoxic conditions. We have also proposed that longevity in the Mit mutants arises as a property of their new metabolic state. If longevity does arise as a function of metabolic state, we would expect to find a common metabolic signature among these animals. To test these predictions, we established a novel approach monitoring the C. elegans exometabolism as a surrogate marker for internal metabolic events. Using HPLC-ultraviolet-based metabolomics and multivariate analyses, we show that long-lived clk-1(qm30) and isp-1(qm150) Mit mutants have a common metabolic profile that is distinct from that of aerobically cultured wild-type animals and, unexpectedly, wild-type animals cultured under severe oxygen deprivation. Moreover, we show that 2 short-lived mitochondrial ETC mutants, mev-1(kn1) and ucr-2.3(pk732), also share a common metabolic signature that is unique. We show that removal of soluble fumarate reductase unexpectedly increases health span in several genetically defined Mit mutants, identifying at least 1 alternate energy production pathway, malate dismutation, that is operative in these animals. Our study suggests long-lived, genetically specified Mit mutants employ a novel metabolism and that life span may well arise as a function of metabolic state.

Artificial small RNA for sequence specific cleavage of target RNA through RNase III endonuclease Dicer

PubMed Central

Liu, Yali; Liu, Li; Zhan, Yonghao; Zhuang, Chengle; Lin, Junhao; Chen, Mingwei; Li, Jianfa; Cai, Zhiming; Huang, Weiren; Zhang, Yong

2016-01-01

CRISPR-Cas9 system uses a guide RNA which functions in conjunction with Cas9 proteins to target a DNA and cleaves double-strand DNA. This phenomenon raises a question whether an artificial small RNA (asRNA), composed of a Dicer–binding RNA element and an antisense RNA, could also be used to induce Dicer to process and degrade a specific RNA. If so, we could develop a new method which is named DICERi for gene silencing or RNA editing. To prove the feasibility of asRNA, we selected MALAT-1 as target and used Hela and MDA-MB-231 cells as experimental models. The results of qRT-PCR showed that the introduction of asRNA decreased the relative expression level of target gene significantly. Next, we analyzed cell proliferation using CCK-8 and EdU staining assays, and then cell migration using wound scratch and Transwell invasion assays. We found that cell proliferation and cell migration were both suppressed remarkably after asRNA was expressed in Hela and MDA-MB-231 cells. Cell apoptosis was also detected through Hoechst staining and ELISA assays and the data indicated that he numbers of apoptotic cell in experimental groups significantly increased compared with negative controls. In order to prove that the gene silencing effects were caused by Dicer, we co-transfected shRNA silencing Dicer and asRNA. The relative expression levels of Dicer and MALAT-1 were both detected and the results indicated that when the cleavage role of Dicer was silenced, the relative expression level of MALAT-1 was not affected after the introduction of asRNA. All the above results demonstrated that these devices directed by Dicer effectively excised target RNA and repressed the target genes, thus causing phenotypic changes. Our works adds a new dimension to gene regulating technologies and may have broad applications in construction of gene circuits. PMID:27231846

Artificial small RNA for sequence specific cleavage of target RNA through RNase III endonuclease Dicer.

PubMed

Xu, Wen; Liu, Yuchen; Liu, Yali; Liu, Li; Zhan, Yonghao; Zhuang, Chengle; Lin, Junhao; Chen, Mingwei; Li, Jianfa; Cai, Zhiming; Huang, Weiren; Zhang, Yong

2016-08-23

CRISPR-Cas9 system uses a guide RNA which functions in conjunction with Cas9 proteins to target a DNA and cleaves double-strand DNA. This phenomenon raises a question whether an artificial small RNA (asRNA), composed of a Dicer-binding RNA element and an antisense RNA, could also be used to induce Dicer to process and degrade a specific RNA. If so, we could develop a new method which is named DICERi for gene silencing or RNA editing. To prove the feasibility of asRNA, we selected MALAT-1 as target and used Hela and MDA-MB-231 cells as experimental models. The results of qRT-PCR showed that the introduction of asRNA decreased the relative expression level of target gene significantly. Next, we analyzed cell proliferation using CCK-8 and EdU staining assays, and then cell migration using wound scratch and Transwell invasion assays. We found that cell proliferation and cell migration were both suppressed remarkably after asRNA was expressed in Hela and MDA-MB-231 cells. Cell apoptosis was also detected through Hoechst staining and ELISA assays and the data indicated that he numbers of apoptotic cell in experimental groups significantly increased compared with negative controls. In order to prove that the gene silencing effects were caused by Dicer, we co-transfected shRNA silencing Dicer and asRNA. The relative expression levels of Dicer and MALAT-1 were both detected and the results indicated that when the cleavage role of Dicer was silenced, the relative expression level of MALAT-1 was not affected after the introduction of asRNA. All the above results demonstrated that these devices directed by Dicer effectively excised target RNA and repressed the target genes, thus causing phenotypic changes. Our works adds a new dimension to gene regulating technologies and may have broad applications in construction of gene circuits.

Intermediates of Krebs cycle correct the depression of the whole body oxygen consumption and lethal cooling in barbiturate poisoning in rat.

PubMed

Ivnitsky, Jury Ju; Schäfer, Timur V; Malakhovsky, Vladimir N; Rejniuk, Vladimir L

2004-10-01

Rats poisoned with one LD50 of thiopental or amytal are shown to increase oxygen consumption when intraperitoneally given sucinate, malate, citrate, alpha-ketoglutarate, dimethylsuccinate or glutamate (the Krebs cycle intermediates or their precursors) but not when given glucose, pyruvate, acetate, benzoate or nicotinate (energy substrates of other metabolic stages etc). Survival was increased with succinate or malate from control groups, which ranged from 30-83% to 87-100%. These effects were unrelated to respiratory depression or hypoxia as judged by little or no effect of succinate on ventilation indices and by the lack of effect of oxygen administration. Body cooling of comatose rats at ambient temperature approximately 19 degrees C became slower with succinate, the rate of cooling correlated well with oxygen consumption decrease. Succinate had no potency to modify oxygen consumption and body temperature in intact rats. A condition for antidote effect of the Krebs intermediate was sufficiently high dosage (5 mmol/kg), further dose increase made no odds. Repeated dosing of succinate had more marked protective effect, than a single one, to oxygen consumption and tended to promote the attenuation of lethal effect of barbiturates. These data suggest that suppression of whole body oxygen consumption with barbiturate overdose could be an important contributor to both body cooling and mortality. Intermediates of Krebs cycle, not only succinate, may have a pronounced therapeutic effect under the proper treatment regimen. Availability of Krebs cycle intermediates may be a limiting factor for the whole body oxygen consumption in barbiturate coma, its role in brain needs further elucidation.

Metabolic engineering of tomato fruit organic acid content guided by biochemical analysis of an introgression line.

PubMed

Morgan, Megan J; Osorio, Sonia; Gehl, Bernadette; Baxter, Charles J; Kruger, Nicholas J; Ratcliffe, R George; Fernie, Alisdair R; Sweetlove, Lee J

2013-01-01

Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). However, the complexity of carboxylic acid metabolism and storage means that it is difficult to predict the best way to engineer altered carboxylic acid levels. Here, we used a biochemical analysis of a tomato introgression line with increased levels of fruit citrate and malate at breaker stage to identify a metabolic engineering target that was subsequently tested in transgenic plants. Increased carboxylic acid levels in introgression line 2-5 were not accompanied by changes in the pattern of carbohydrate oxidation by pericarp discs or the catalytic capacity of tricarboxylic acid cycle enzymes measured in isolated mitochondria. However, there was a significant decrease in the maximum catalytic activity of aconitase in total tissue extracts, suggesting that a cytosolic isoform of aconitase was affected. To test the role of cytosolic aconitase in controlling fruit citrate levels, we analyzed fruit of transgenic lines expressing an antisense construct against SlAco3b, one of the two tomato genes encoding aconitase. A green fluorescent protein fusion of SlAco3b was dual targeted to cytosol and mitochondria, while the other aconitase, SlAco3a, was exclusively mitochondrial when transiently expressed in tobacco (Nicotiana tabacum) leaves. Both aconitase transcripts were decreased in fruit from transgenic lines, and aconitase activity was reduced by about 30% in the transgenic lines. Other measured enzymes of carboxylic acid metabolism were not significantly altered. Both citrate and malate levels were increased in ripe fruit of the transgenic plants, and as a consequence, total carboxylic acid content was increased by 50% at maturity.

Metabolic Engineering of Tomato Fruit Organic Acid Content Guided by Biochemical Analysis of an Introgression Line1[W][OA

PubMed Central

Morgan, Megan J.; Osorio, Sonia; Gehl, Bernadette; Baxter, Charles J.; Kruger, Nicholas J.; Ratcliffe, R. George; Fernie, Alisdair R.; Sweetlove, Lee J.

2013-01-01

Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). However, the complexity of carboxylic acid metabolism and storage means that it is difficult to predict the best way to engineer altered carboxylic acid levels. Here, we used a biochemical analysis of a tomato introgression line with increased levels of fruit citrate and malate at breaker stage to identify a metabolic engineering target that was subsequently tested in transgenic plants. Increased carboxylic acid levels in introgression line 2-5 were not accompanied by changes in the pattern of carbohydrate oxidation by pericarp discs or the catalytic capacity of tricarboxylic acid cycle enzymes measured in isolated mitochondria. However, there was a significant decrease in the maximum catalytic activity of aconitase in total tissue extracts, suggesting that a cytosolic isoform of aconitase was affected. To test the role of cytosolic aconitase in controlling fruit citrate levels, we analyzed fruit of transgenic lines expressing an antisense construct against SlAco3b, one of the two tomato genes encoding aconitase. A green fluorescent protein fusion of SlAco3b was dual targeted to cytosol and mitochondria, while the other aconitase, SlAco3a, was exclusively mitochondrial when transiently expressed in tobacco (Nicotiana tabacum) leaves. Both aconitase transcripts were decreased in fruit from transgenic lines, and aconitase activity was reduced by about 30% in the transgenic lines. Other measured enzymes of carboxylic acid metabolism were not significantly altered. Both citrate and malate levels were increased in ripe fruit of the transgenic plants, and as a consequence, total carboxylic acid content was increased by 50% at maturity. PMID:23166354

Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons.

PubMed

Llorente-Folch, Irene; Rueda, Carlos B; Amigo, Ignacio; del Arco, Araceli; Saheki, Takeyori; Pardo, Beatriz; Satrústegui, Jorgina

2013-08-28

Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.

Absolute configuration of a chiral CHD group via neutron diffraction: confirmation of the absolute stereochemistry of the enzymatic formation of malic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bau, R.; Brewer, I.; Chiang, M.Y.

Neutron diffraction has been used to monitor the absolute stereochemistry of an enzymatic reaction. (-)(2S)malic-3-d acid was prepared by the action of fumarase on fumaric acid in D/sub 2/O. After a large number of cations were screened, it was found that (+)(R)..cap alpha..-phenylethylamine forms the large crystals necessary for a neutron diffraction analysis. The subsequent structure determination showed that (+)(R)..cap alpha..-phenylethylammonium (-)(2S)malate-3-d has an absolute configuration of R at the CHD site. This result confirms the absolute stereochemistry of fumarate-to-malate transformation as catalyzed by the enzyme fumarase.

Nocturnal Accumulation of Malic Acid Occurs in Mesophyll Tissue without Proton Transport to Epidermal Tissue in the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum1

PubMed Central

Winter, Klaus; Edwards, Gerald E.; Holtum, Joseph A. M.

1981-01-01

The inducible Crassulacean acid metabolism plant, Mesembryanthemum crystallinum, accumulates malic acid, i.e. equivalent amounts of malate anions and protons in the mesophyll cells at night. Levels of malate and titratable acidity are low in the epidermal tissue and do not change significantly during the day/night cycle. This result is in contrast to a recent report (Bloom 1979 Plant Physiol 64: 919-923) that the synthesis of malic acid during dark CO2 fixation is associated with an equivalent exchange of inorganic cations from epidermal tissue with protons in the mesophyll cells. PMID:16661916

Rat liver mitochondrial dysfunction by addition of copper(II) or iron(III) ions.

PubMed

Saporito-Magriñá, Christian; Musacco-Sebio, Rosario; Acosta, Juan M; Bajicoff, Sofía; Paredes-Fleitas, Paola; Boveris, Alberto; Repetto, Marisa G

2017-01-01

Increased copper (Cu) and iron (Fe) levels in liver and brain are associated to oxidative stress and damage with increased phospholipid oxidation process. The aim of this work was to assess the toxic effects of Cu 2+ and Fe 3+ addition to rat liver mitochondria by determining mitochondrial respiration in states 3 (active respiration) and 4 (resting respiration), and phospholipid peroxidation. Both, Cu 2+ and Fe 3+ produced decreases in O 2 consumption in a concentration-dependent manner in active state 3: both ions by 42% with malate-glutamate as complex I substrate (concentration for half maximal response (C 50 ) 60μM Cu 2+ and 1.25mM Fe 3+ ), and with succinate as complex II substrate: 64-69% with C 50 of 50μM Cu 2+ and with C 50 of 1.25mM of Fe 3+ . Respiratory control decreased with Cu 2+ (C 50 50μM) and Fe 3+ (C 50 1.25-1-75mM) with both substrates. Cu 2+ produced a 2-fold increase and Fe 3+ a 5-fold increase of thiobarbituric acid-reactive substances (TBARS) content from 25μM Cu 2+ (C 50 40μM) and from 100μM Fe 3+ (C 50 1.75mM). Supplementations with Cu 2+ and Fe 3+ ions induce mitochondrial dysfunction with phospholipid peroxidation in rat liver mitochondria. Although is proved that a Fenton/Haber Weiss mechanism of oxidative damage occurs in metal-ion induced mitochondrial toxicity, slightly different responses to the metal ions suggest some differences in the mechanism of intracellular toxicity. The decreased rates of mitochondrial respiration and the alteration of mitochondrial function by phospholipid and protein oxidations lead to mitochondrial dysfunction, cellular dyshomeostasis and cell death. Copyright © 2016 Elsevier Inc. All rights reserved.

Light versus Dark Carbon Metabolism in Cherry Tomato Fruits

PubMed Central

Laval-Martin, Danielle; Farineau, Jack; Diamond, Jeffrey

1977-01-01

The photosynthetic properties of the internal and peripheral tissues of the cherry tomato fruit (Lycopersicum esculentum var. cerasiforme Dun A. Gray) were investigated. Whole fruit and their isolated tissues evolve large amounts of CO2 in darkness. In the light, this evolution decreases but nevertheless remains a net evolution; 3-(3,4-dichlorophenyl)-1,1-dimethylurea abolishes the effects of light. Incorporation of 14CO2 by leaves and fruit tissues demonstrates that the outer region of the fruit has the highest photosynthetic efficiency on a chlorophyll basis; the internal fruit tissue, richer in chlorophyll, has a much lower efficiency. The identification of intermediates following short term incubations with 14CO2 shows that in darkness the fruit accumulates the majority of label in malate. In the light, leaf tissue exhibits a pattern of incorporation characteristic of C-3 metabolism, whereas fruit tissue exhibits a decreased labeling of malate with a concomitant appearance of label in Calvin cycle intermediates. This is in agreement with the levels and types of carboxylating activities demonstrated in vitro; especially noteworthy is the very low ribulose diphosphate carboxylase activity in the internal fruit tissue. The photosynthetic potential, phosphoenolpyruvate carboxylase activity, and quantities of malate accumulated by fruit tissues are parallel to their chlorophyll content during growth and maturation. PMID:16660204

Compartmentation Studies on Spinach Leaf Peroxisomes 1

PubMed Central

Heupel, Ralf; Markgraf, Therese; Robinson, David G.; Heldt, Hans Walter

1991-01-01

In concurrence with earlier results, the following enzymes showed latency in intact spinach (Spinacia oleracea L.) leaf peroxisomes: malate dehydrogenase (89%), hydroxypyruvate reductase (85%), serine glyoxylate aminotransferase (75%), glutamate glyoxylate aminotransferase (41%), and catalase (70%). In contrast, glycolate oxidase was not latent. Aging of peroxisomes for several hours resulted in a reduction in latency accompanied by a partial solubilization of the above mentioned enzymes. The extent of enzyme solubilization was different, being highest with glutamate glyoxylate aminotransferase and lowest with malate dehydrogenase. Osmotic shock resulted in only a partial reduction of enzyme latency. Electron microscopy revealed that the osmotically shocked peroxisomes remained compact, with smaller particle size and pleomorphic morphology but without a continuous boundary membrane. Neither in intact nor in osmotically shocked peroxisomes was a lag phase observed in the formation of glycerate upon the addition of glycolate, serine, malate, and NAD. Apparently, the intermediates, glyoxylate, hydroxypyruvate, and NADH, were confined within the peroxisomal matrix in such a way that they did not readily leak out into the surrounding medium. We conclude that the observed compartmentation of peroxisomal metabolism is not due to the peroxisomal boundary membrane as a permeability barrier, but is a function of the structural arrangement of enzymes in the peroxisomal matrix allowing metabolite channeling. ImagesFigure 3 PMID:16668283

Analysis of the Mycoplasma bovis lactate dehydrogenase reveals typical enzymatic activity despite the presence of an atypical catalytic site motif.

PubMed

Masukagami, Yumiko; Tivendale, Kelly Anne; Browning, Glenn Francis; Sansom, Fiona Margaret

2018-02-01

The lactate dehydrogenase (LDH) of Mycoplasma genitalium has been predicted to also act as a malate dehydrogenase (MDH), but there has been no experimental validation of this hypothesized dual function for any mollicute. Our analysis of the metabolite profile of Mycoplasma bovis using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) detected malate, suggesting that there may be MDH activity in M. bovis. To investigate whether the putative l-LDH enzyme of M. bovis has a dual function (MDH and LDH), we performed bioinformatic and functional biochemical analyses. Although the amino acid sequence and predicted structural analysis of M. bovisl-LDH revealed unusual residues within the catalytic site, suggesting that it may have the flexibility to possess a dual function, our biochemical studies using recombinant M. bovis -LDH did not detect any MDH activity. However, we did show that the enzyme has typical LDH activity that could be inhibited by both MDH substrates oxaloacetate (OAA) and malate, suggesting that these substrates may be able to bind to M. bovis LDH. Inhibition of the conversion of pyruvate to lactate by OAA may be one method the mycoplasma cell uses to reduce the potential for accumulation of intracellular lactate.

Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

PubMed

Abbrescia, Daniela Isabel; La Piana, Gianluigi; Lofrumento, Nicola Elio

2012-02-15

In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase. Copyright © 2011 Elsevier Inc. All rights reserved.

Interplant Aboveground Signaling Prompts Upregulation of Auxin Promoter and Malate Transporter as Part of Defensive Response in the Neighboring Plants.

PubMed

Sweeney, Connor; Lakshmanan, Venkatachalam; Bais, Harsh P

2017-01-01

When disrupted by stimuli such as herbivory, pathogenic infection, or mechanical wounding, plants secrete signals such as root exudates and volatile organic compounds (VOCs). The emission of VOCs induces a response in the neighboring plant communities and can improve plant fitness by alerting nearby plants of an impending threat and prompting them to alter their physiology for defensive purposes. In this study, we investigated the role of plant-derived signals, released as a result of mechanical wounding, that may play a role in intraspecific communication between Arabidopsis thaliana communities. Plant-derived signals released by the wounded plant resulted in more elaborate root development in the neighboring, unwounded plants. Such plant-derived signals also upregulated the Aluminum-activated malate transporter ( ALMT1 ) responsible for the secretion of malic acid (MA) and the DR5 promoter, an auxin responsive promoter concentrated in root apex of the neighboring plants. We speculate that plant-derived signal-induced upregulation of root-specific ALMT1 in the undamaged neighboring plants sharing the environment with stressed plants may associate more with the benign microbes belowground. We also observed increased association of beneficial bacterium Bacillus subtilis UD1022 on roots of the neighboring plants sharing environment with the damaged plants. Wounding-induced plant-derived signals therefore induce defense mechanisms in the undamaged, local plants, eliciting a two-pronged preemptive response of more rapid root growth and up-regulation of ALMT1 , resulting in increased association with beneficial microbiome.

Imbalanced lignin biosynthesis promotes the sexual reproduction of homothallic oomycete pathogens.

PubMed

Quentin, Michaël; Allasia, Valérie; Pegard, Anthony; Allais, Florent; Ducrot, Paul-Henri; Favery, Bruno; Levis, Caroline; Martinet, Sophie; Masur, Clarissa; Ponchet, Michel; Roby, Dominique; Schlaich, Nikolaus L; Jouanin, Lise; Keller, Harald

2009-01-01

Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-L-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-L-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment.

Imbalanced Lignin Biosynthesis Promotes the Sexual Reproduction of Homothallic Oomycete Pathogens

PubMed Central

Quentin, Michaël; Allasia, Valérie; Pegard, Anthony; Allais, Florent; Ducrot, Paul-Henri; Favery, Bruno; Levis, Caroline; Martinet, Sophie; Masur, Clarissa; Ponchet, Michel; Roby, Dominique; Schlaich, Nikolaus L.; Jouanin, Lise; Keller, Harald

2009-01-01

Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-l-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-l-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment. PMID:19148278

Accumulation of ammonium in Norway spruce (Picea abies) seedlings measured by in vivo 14N-NMR.

PubMed

Aarnes, H; Eriksen, A B; Petersen, D; Rise, F

2007-01-01

(14)N-NMR and (31)P-NMR have been used to monitor the in vivo pH in roots, stems, and needles from seedlings of Norway spruce, a typical ammonium-tolerant plant. The vacuolar and cytoplasmic pH measured by (31)P-NMR was found to be c. pH 4.8 and 7.0, respectively, with no significant difference between plants growing with ammonium or nitrate as the N-source. The (1)H-coupled (14) NH 4+ resonance is pH-sensitive: at alkaline pH it is a narrow singlet line and below pH 4 it is an increasing multiplet line with five signals. The pH values in ammonium-containing compartments measured by (14)N-NMR ranged from 3.7 to 3.9, notably lower than the estimated pH values of the P(i) pools. This suggests that, in seedlings of Norway spruce, ammonium is stored in vacuoles with low pH possibly to protect the seedlings against the toxic effects of ammonium ( NH 4+) or ammonia (NH3). It was also found that concentrations of malate were 3-6 times higher in stems than in roots and needles, with nitrate-grown plants containing more malate than plants grown with ammonium.

Immunological response and protection of mice immunized with plasmid encoding Toxoplasma gondii glycolytic enzyme malate dehydrogenase.

PubMed

Hassan, I A; Wang, S; Xu, L; Yan, R; Song, X; XiangRui, L

2014-12-01

Toxoplasma gondii Malate dehydrogenase (TgMDH) plays an important role as part of the energy production cycle. In this investigation, immunological changes and protection efficiency of this protein delivered as a DNA vaccine have been evaluated. Mice were intramuscularly immunized with pTgMDH, followed by challenge with virulent T. gondii RH strain, 2 weeks after the booster immunization. Compared to the control groups, the results showed that pTgMDH has stimulated specific humoral response as demonstrated by significant high titers of total IgG and subclasses IgG1 and IgG2a , beside IgA and IgM, but not IgE. Analysis of cytokine profiles revealed significant increases of IFN-γ, IL-4 and IL-17, while no significant changes were detected in TGF-β1. In cell-mediated response, both T lymphocytes subpopulations CD4(+) and CD8(+) were positively recruited as significant percentages were recorded in response to immunization with TgMDH. Significant long survival rate, 17 days, has been observed in the TgMDH vaccinated group, in contrast with control groups which died within 8-9 days after challenge. These results demonstrated that TgMDH could induce significant immunological responses leading to a considerable level of protection against acute toxoplasmosis infection. © 2014 John Wiley & Sons Ltd.

Molecular characterization and functional analysis of Eimeria tenella malate dehydrogenase.

PubMed

Chen, Ting; Huang, Bing; Zhao, Qiping; Dong, Hui; Zhu, Shunhai; Zhao, Zongping; Lv, Ling; Yan, Ming; Han, Hongyu

2018-05-08

Eimeria tenella is a serious intracellular parasite that actively invades cecal epithelial cells of chickens. The widespread use of drugs causes severe resistance to Eimeria tenella. We detected that malate dehydrogenase (MDH), one of the differentially expressed genes, was upregulated in diclazuril-resistant and maduramicin-resistant strains through transcriptome sequencing. In this study, we cloned and expressed MDH of E. tenella (EtMDH). Quantitative real-time polymerase chain reactions (qPCR) and Western blots were used to analyze the expression of EtMDH in resistant and sensitive strains, indicating EtMDH was upregulated in two resistant strains at the messenger RNA and protein levels. Enzyme activity was tested through absorbance measurement and the EtMDH activity increased in two resistant strains. Expression levels of EtMDH in four developmental stages of E. tenella were tested through qPCR and Western blot. Invasion inhibition assays explored if EtMDH was involved in invasion of DF-1 cells by E. tenella sporozoites. Indirect immunofluorescence assays investigated EtMDH distribution during parasite development in DF-1 cells invaded by E. tenella sporozoites. Experimental results showed that EtMDH may be related to drug resistance of E. tenella during its development and invasion. EtMDH may be an effective molecular marker for detection of E. tenella drug resistance.

The ALMT Family of Organic Acid Transporters in Plants and Their Involvement in Detoxification and Nutrient Security.

PubMed

Sharma, Tripti; Dreyer, Ingo; Kochian, Leon; Piñeros, Miguel A

2016-01-01

About a decade ago, members of a new protein family of anion channels were discovered on the basis of their ability to confer on plants the tolerance toward toxic aluminum ions in the soil. The efflux of Al 3+ -chelating malate anions through these channels is stimulated by external Al 3+ ions. This feature of a few proteins determined the name of the entire protein family as Aluminum-activated Malate Transporters (ALMT). Meanwhile, after several years of research, it is known that the physiological roles of ALMTs go far beyond Al-detoxification. In this review article we summarize the current knowledge on this transporter family and assess their involvement in diverse physiological processes.

The ALMT Family of Organic Acid Transporters in Plants and Their Involvement in Detoxification and Nutrient Security

PubMed Central

Sharma, Tripti; Dreyer, Ingo; Kochian, Leon; Piñeros, Miguel A.

2016-01-01

About a decade ago, members of a new protein family of anion channels were discovered on the basis of their ability to confer on plants the tolerance toward toxic aluminum ions in the soil. The efflux of Al3+-chelating malate anions through these channels is stimulated by external Al3+ ions. This feature of a few proteins determined the name of the entire protein family as Aluminum-activated Malate Transporters (ALMT). Meanwhile, after several years of research, it is known that the physiological roles of ALMTs go far beyond Al-detoxification. In this review article we summarize the current knowledge on this transporter family and assess their involvement in diverse physiological processes. PMID:27757118

Selection of Streptococcus lactis Mutants Defective in Malolactic Fermentation

PubMed Central

Renault, Pierre P.; Heslot, Henri

1987-01-01

An enrichment medium and a new sensitive medium were developed to detect malolactic variants in different strains of lactic bacteria. Factors such as the concentration of glucose and l-malate, pH level, and the type of indicator dye used are discussed with regard to the kinetics of malic acid conversion to lactic acid. Use of these media allowed a rapid and easier screening of mutagenized streptococcal cells unable to ferment l-malate. A collection of malolactic-negative mutants of Streptococcus lactis induced by UV, nitrosoguanidine, or transposonal mutagenesis were characterized. The results showed that several mutants were apparently defective in the structural gene of malolactic enzyme, whereas others contained mutations which may either inactivate a putative permease or affect a regulatory sequence. PMID:16347282

Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia

PubMed Central

Ma, Zuheng; Scholz, Hanne; Björklund, Anneli; Grill, Valdemar

2015-01-01

Objective To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia. Methods and Design Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20–22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. Results Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. Conclusions Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting. Results also indicate idiosyncrasies of β-cells that do not respire in response to a standard inclusion of malate in SUIT protocols. PMID:26401848

Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia.

PubMed

Hals, Ingrid K; Bruerberg, Simon Gustafson; Ma, Zuheng; Scholz, Hanne; Björklund, Anneli; Grill, Valdemar

2015-01-01

To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia. Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20-22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting. Results also indicate idiosyncrasies of β-cells that do not respire in response to a standard inclusion of malate in SUIT protocols.

Physiological role of AOX1a in photosynthesis and maintenance of cellular redox homeostasis under high light in Arabidopsis thaliana.

PubMed

Vishwakarma, Abhaypratap; Bashyam, Leena; Senthilkumaran, Balasubramanian; Scheibe, Renate; Padmasree, Kollipara

2014-08-01

As plants are sessile, they often face high light (HL) stress that causes damage of the photosynthetic machinery leading to decreased photosynthesis. The importance of alternative oxidase (AOX) in optimizing photosynthesis is well documented. In the present study, the role of AOX in sustaining photosynthesis under HL was studied using AOX1a knockout mutants (aox1a) of Arabidopsis thaliana. Under growth light (GL; 50 μmol photons m(-2) s(-1)) conditions, aox1a plants did not show any changes in photosynthetic parameters, NAD(P)/H redox ratios, or respiratory O2 uptake when compared to wild-type (WT). Upon exposure to HL (700 μmol photons m(-2) s(-1)), respiratory rates did not vary between WT and aox1a. But, photosynthetic parameters related to photosystem II (PSII) and NaHCO3 dependent O2 evolution decreased, while the P700 reduction state increased in aox1a compared to WT. Further, under HL, the redox state of cellular NAD(P)/H pools increased with concomitant rise in reactive oxygen species (ROS) and malondialdehyde (MDA) content in aox1a compared to WT. In presence of HL, the transcript levels of several genes related to antioxidant, malate-oxaloacetate (malate-OAA) shuttle, photorespiratory and respiratory enzymes was higher in aox1a compared to WT. Taken together, these results demonstrate that under HL, in spite of significant increase in transcript levels of several genes mentioned above to maintain cellular redox homeostasis and minimize ROS production, Arabidopsis plants deficient in AOX1a were unable to sustain photosynthesis as is the case in WT plants. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Systematic biology analysis on photosynthetic carbon metabolism of maize leaf following sudden heat shock under elevated CO2.

PubMed

Qu, Mingnan; Chen, Genyun; Bunce, James A; Zhu, Xiaocen; Sicher, Richard C

2018-05-18

Plants would experience more complex environments, such as sudden heat shock (SHS) stress combined with elevated CO 2 in the future, and might adapt to this stressful condition by optimizing photosynthetic carbon metabolism (PCM). It is interesting to understand whether this acclimation process would be altered in different genotypes of maize under elevated CO 2 , and which metabolites represent key indicators reflecting the photosynthetic rates (P N ) following SHS. Although B76 had greater reduction in P N during SHS treatment, our results indicated that P N in genotype B76, displayed faster recovery after SHS treatment under elevated CO 2 than in genotype B106. Furthermore, we employed a stepwise feature extraction approach by partial linear regression model. Our findings demonstrated that 9 key metabolites over the total (35 metabolites) can largely explain the variance of P N during recovery from SHS across two maize genotypes and two CO 2 grown conditions. Of these key metabolites, malate, valine, isoleucine, glucose and starch are positively correlated with recovery pattern of P N . Malate metabolites responses to SHS were further discussed by incorporating with the activities and gene expression of three C 4 photosynthesis-related key enzymes. We highlighted the importance of malate metabolism during photosynthesis recovery from short-term SHS, and data integration analysis to better comprehend the regulatory framework of PCM in response to abiotic stress.

A Dicarboxylate Transporter, LjALMT4, Mainly Expressed in Nodules of Lotus japonicus.

PubMed

Takanashi, Kojiro; Sasaki, Takayuki; Kan, Tomohiro; Saida, Yuka; Sugiyama, Akifumi; Yamamoto, Yoko; Yazaki, Kazufumi

2016-07-01

Legume plants can establish symbiosis with soil bacteria called rhizobia to obtain nitrogen as a nutrient directly from atmospheric N2 via symbiotic nitrogen fixation. Legumes and rhizobia form nodules, symbiotic organs in which fixed-nitrogen and photosynthetic products are exchanged between rhizobia and plant cells. The photosynthetic products supplied to rhizobia are thought to be dicarboxylates but little is known about the movement of dicarboxylates in the nodules. In terms of dicarboxylate transporters, an aluminum-activated malate transporter (ALMT) family is a strong candidate responsible for the membrane transport of carboxylates in nodules. Among the seven ALMT genes in the Lotus japonicus genome, only one, LjALMT4, shows a high expression in the nodules. LjALMT4 showed transport activity in a Xenopus oocyte system, with LjALMT4 mediating the efflux of dicarboxylates including malate, succinate, and fumarate, but not tricarboxylates such as citrate. LjALMT4 also mediated the influx of several inorganic anions. Organ-specific gene expression analysis showed LjALMT4 mRNA mainly in the parenchyma cells of nodule vascular bundles. These results suggest that LjALMT4 may not be involved in the direct supply of dicarboxylates to rhizobia in infected cells but is responsible for supplying malate as well as several anions necessary for symbiotic nitrogen fixation, via nodule vasculatures.

Availability of neurotransmitter glutamate is diminished when beta-hydroxybutyrate replaces glucose in cultured neurons.

PubMed

Lund, Trine M; Risa, Oystein; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

2009-07-01

Ketone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4-(13)C]beta-hydroxybutyrate to that of [1,6-(13)C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate-glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of (13)C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4-(13)C]beta-hydroxybutyrate as opposed to [1,6-(13)C]glucose. Our results suggest that the change in aspartate-glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate-aspartate shuttle activity in neurons using beta-hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only beta-hydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing beta-hydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate-aspartate shuttle.

Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2

PubMed Central

Everett, Peter; Clish, Clary B.; Sukhatme, Vikas P.

2010-01-01

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for leukemia therapy. PMID:20824065

Proteotoxicity and the contrasting effects of oxaloacetate and glycerol on Caenorhabditis elegans life span: a role for methylglyoxal?

PubMed

Hipkiss, Alan R

2010-10-01

Because accumulation of altered proteins is the most common biochemical symptom of aging, it is at least possible that such proteotoxicity may cause aging and influence life span. The life span of the nematode worm Caenorhabditis elegans is strongly influenced by changes in the intracellular concentration of methylglyoxal (MG), a putative source of much age-related proteotoxicity and organelle, cellular, and molecular dysfunction. Glycerol has recently been shown to shorten, whereas oxaloacetate has been found to extend, life span in C. elegans. It is suggested here that glycerol and oxaloacetate exert opposing effects on MG formation in C. elegans. It is proposed that, if not secreted by aquaporin, glycerol is converted to glycerol phosphate and then to dihydroxyacetone phosphate (DHAP) via a reaction requiring nicotinamide adenine dinucleotide (NAD(+)). This inhibits operation of the glycerol phosphate cycle in which DHAP is converted into glycerol phosphate, which concomitantly regenerates NAD(+) from NADH, thereby ensuring glycolytic oxidation of glyceraldehyde-3-phosphate (G3P). Because DHAP and G3P spontaneously decompose into MG, and NAD(+) is required for conversion of G3P into phosphoglycerate, the glycerol-induced increased DHAP formation and decreased NAD(+) availability will increase the potential for MG generation. In contrast, oxaloacetate may decrease MG generation by stimulating the operation of the malate-oxaloacetate shuttle, in which oxaloacetate is converted to malate, which regenerates NAD(+) from NADH. By the ensuing G3P oxidation, increased NAD(+) availability will decrease the potential for MG formation. It should be noted that mitochondria are involved in the operation of the above cycle/shuttles and that increased NAD(+) availability also stimulates those sirtuin activities that increase mitogenesis and mitochondrial activity via effects on signal transduction and gene expression, which frequently accompany dietary restriction-induced life span extension.

Testosterone and muscle hypertrophy in female rats

NASA Technical Reports Server (NTRS)

Kuhn, F. E.; Max, S. R.

1985-01-01

The effects of chronic treatment with testosterone propionate (TP) on compensatory muscle hypertropy in female rats are examined. The 48 female rats were placed in one of four test groups: (1) no overload (synergist removal), no TP, (2) overload, no TP, (3) no overload + TP, and (4) overload + TP. The technique used to administer the TP is described. The preparation of the plantaris muscle, the analysis of pyruvate oxidation and the determination of malate and lactate dehydrogenases and the noncollogen protein are explained. The results which reveal the effect of overload and TP on body weight, noncollogen protein concentration, lactate and malate dehydrogenase activities, and pyruvate oxidation are presented and discussed. It is concluded that in terms of body weight, protein content, pyruvate, glycolysis, and oxidative metabolisms chronic TP treatments do not change compensatory muscle hypertropy.

Chitosan nanoencapsulated exogenous trypsin biomimics zymogen-like enzyme in fish gastrointestinal tract.

PubMed

Kumari, Rakhi; Gupta, Subodh; Singh, Arvind R; Ferosekhan, S; Kothari, Dushyant C; Pal, Asim Kumar; Jadhao, Sanjay Balkrishna

2013-01-01

Exogenous proteolytic enzyme supplementation is required in certain disease conditions in humans and animals and due to compelling reasons on use of more plant protein ingredients and profitability in animal feed industry. However, limitations on their utility in diet are imposed by their pH specificity, thermolabile nature, inhibition due to a variety of factors and the possibility of intestinal damage. For enhancing the efficacy and safety of exogenous trypsin, an efficient chitosan (0.04%) nanoencapsulation-based controlled delivery system was developed. An experiment was conducted for 45 days to evaluate nanoencapsulated trypsin (0.01% and 0.02%) along with 0.02% bare trypsin and 0.4% chitosan nanoparticles against a control diet on productive efficiency (growth rate, feed conversion and protein efficiency ratio), organo-somatic indices, nutrient digestibility, tissue enzyme activities, hematic parameters and intestinal histology of the fish Labeo rohita. All the synthesized nanoparticles were of desired characteristics. Enhanced fish productive efficiency using nanoencapsulated trypsin over its bare form was noticed, which corresponded with enhanced (P<0.01) nutrient digestibility, activity of intestinal protease, liver and muscle tissue transaminases (alanine and aspartate) and dehydrogenases (lactate and malate), serum blood urea nitrogen and serum protein profile. Intestinal tissues of fish fed with 0.02% bare trypsin showed broadened, marked foamy cells with lipid vacuoles. However, villi were healthier in appearance with improved morphological features in fish fed with nanoencapsulated trypsin than with bare trypsin, and the villi were longer in fish fed with 0.01% nanoencapsulated trypsin than with 0.02% nanoencapsulated trypsin. The result of this premier experiment shows that nanoencapsulated trypsin mimics zymogen-like proteolytic activity via controlled release, and hence the use of 0.01% nanoencapsulated trypsin (in chitosan nanoparticles) over bare trypsin can be favored as a dietary supplement in animals and humans.

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase.

PubMed

Miginiac-Maslow, M; Jacquot, J P; Droux, M

1985-09-01

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxinm and f (Tdm, Tdf) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m(-2) under nitrogen and 50 W·m(-2) under air, while NADP photoreduction was saturated at 240 W·m(-2). Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N2 by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O2 was located at the level of Fd.

Remarkable Reproducibility of Enzyme Activity Profiles in Tomato Fruits Grown under Contrasting Environments Provides a Roadmap for Studies of Fruit Metabolism1[W][OPEN

PubMed Central

Biais, Benoît; Bénard, Camille; Beauvoit, Bertrand; Colombié, Sophie; Prodhomme, Duyên; Ménard, Guillaume; Bernillon, Stéphane; Gehl, Bernadette; Gautier, Hélène; Ballias, Patricia; Mazat, Jean-Pierre; Sweetlove, Lee; Génard, Michel; Gibon, Yves

2014-01-01

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum ‘Moneymaker’) plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels. PMID:24474652

Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

PubMed Central

2010-01-01

Background Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications. Results A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from Bacteroides sp. 2_1_33B and Parabacteroides distasonis ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg2+. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: Km= 0.48 mM, Vmax = 827 μM/min/mg, and kcat/Km = 1900 mM/s. Conclusions We isolated a novel fumarase gene, fumF, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of L-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules. PMID:21092234

Oxidative damage of mitochondrial proteins contributes to fruit senescence: a redox proteomics analysis.

PubMed

Qin, Guozheng; Meng, Xianghong; Wang, Qing; Tian, Shiping

2009-05-01

Oxidative damage to mitochondria caused by reactive oxygen species (ROS) has been implicated in the process of senescence as well as a number of senescence-related disorders in a variety of organisms. Whereas mitochondrial DNA was shown to be oxidatively modified during cellular senescence, mitochondrial protein oxidation is not well-understood. With the use of high-resolution, two-dimensional gel electrophoresis coupled with immunoblotting, we show here that protein carbonylation, a widely used marker of protein oxidation, increased in mitochondria during the senescence of peach fruit. Specific mitochondrial proteins including outer membrane transporter (voltage-dependent anion-selective channel, VDAC), tricarboxylic acid cycle enzymes (malate dehydrogenase and aconitase), and antioxidant proteins (manganese superoxide dismutase, MnSOD) were found as the targets. The oxidative modification was concomitant with a change of VDAC function and loss of catalytic activity of malate dehydrogenase and MnSOD, which in turn facilitated the release of superoxide radicals in mitochondria. Reduction of ROS content by lowering the environmental temperature prevented the accumulation of protein carbonylation in mitochondria and retarded fruit senescence, whereas treatment of fruit with H2O2 had the opposite effect. Our data suggest that oxidative damage of specific mitochondrial proteins may be responsible for impairment of mitochondrial function, thus, leading to fruit senescence. Proteomics analysis of mitochondrial redox proteins provides considerable information on the molecular mechanisms involved in the progression of fruit senescence.

Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux.

PubMed

Ligaba, Ayalew; Maron, Lyza; Shaff, Jon; Kochian, Leon; Piñeros, Miguel

2012-07-01

Root efflux of organic acid anions underlies a major mechanism of plant aluminium (Al) tolerance on acid soils. This efflux is mediated by transporters of the Al-activated malate transporter (ALMT) or the multi-drug and toxin extrusion (MATE) families. ZmALMT2 was previously suggested to be involved in Al tolerance based on joint association-linkage mapping for maize Al tolerance. In the current study, we functionally characterized ZmALMT2 by heterologously expressing it in Xenopus laevis oocytes and transgenic Arabidopsis. In oocytes, ZmALMT2 mediated an Al-independent electrogenic transport product of organic and inorganic anion efflux. Ectopic overexpression of ZmALMT2 in an Al-hypersensitive Arabidopsis KO/KD line lacking the Al tolerance genes, AtALMT1 and AtMATE, resulted in Al-independent constitutive root malate efflux which partially restored the Al tolerance phenotype. The lack of correlation between ZmALMT2 expression and Al tolerance (e.g., expression not localized to the root tip, not up-regulated by Al, and higher in sensitive versus tolerance maize lines) also led us to question ZmALMT2's role in Al tolerance. The functional properties of the ZmALMT2 transporter presented here, along with the gene expression data, suggest that ZmALMT2 is not involved in maize Al tolerance but, rather, may play a role in mineral nutrient acquisition and transport. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.

Development of antimigraine transdermal delivery systems of pizotifen malate.

PubMed

Serna-Jiménez, C E; del Rio-Sancho, S; Calatayud-Pascual, M A; Balaguer-Fernández, C; Femenía-Font, A; López-Castellano, A; Merino, V

2015-08-15

The aim of this study was to develop and evaluate a transdermal delivery system of pizotifen malate. Pizotifen is frequently used in the preventive treatment of migraine, but is also indicated in eating disorders. In the course of the project, the effects of chemical enhancers such as ethanol, 1,8-cineole, limonene, azone and different fatty acids (decanoic, decenoic, dodecanoic, linoleic and oleic acids) were determined, first using a pizotifen solution. Steady state flux, diffusion and partition parameters were estimated by fitting the Scheuplein equation to the data obtained. Among the chemical enhancers studied, decenoic acid showed the highest enhancement activity, which seemed to be due to the length of its alkyl chain and unsaturation at the 9th carbon. The influence of iontophoresis and the involvement of electrotransport in said process was determined. The absorption profile obtained with iontophoresis was similar to that obtained with fatty acids and terpenes, though skin deposition of the drug was lower with the former. Transdermal delivery systems (TDS) of pizotifen were manufactured by including chemical enhancers, decenoic acid or oleic acid, and were subsequently characterized. When the results obtained with solutions were compared with those obtained with the TDS, a positive enhancement effect was observed with the latter with respect to the partitioning and diffusion of the drug across the skin. Our findings endorse the suitability of our TDS for delivering therapeutic amounts of pizotifen malate. Copyright © 2015 Elsevier B.V. All rights reserved.

Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Decottignies, P.; Schmitter, J.M.; Miginiac-Maslow, M.

1988-08-25

The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by (14C)iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two (14C)carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, themore » analysis of the tryptic digest of light-activated (14C)carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.« less

Increasing fatty acid production in E. coli by simulating the lipid accumulation of oleaginous microorganisms.

PubMed

Meng, Xin; Yang, Jianming; Cao, Yujin; Li, Liangzhi; Jiang, Xinglin; Xu, Xin; Liu, Wei; Xian, Mo; Zhang, Yingwei

2011-08-01

Unlike many oleaginous microorganisms, E. coli only maintains a small amount of natural lipids in cells, impeding its utility to overproduce fatty acids. In this study, acetyl-CoA carboxylase (ACC) from Acinetobacter calcoaceticus was expressed in E. coli to redirect the carbon flux to the generation of malonyl-CoA, which resulted in a threefold increase in intracellular lipids. Moreover, providing a high level of NADPH by overexpressing malic enzyme and adding malate to the culture medium resulted in a fourfold increase in intracellular lipids (about 197.74 mg/g). Co-expression of ACC and malic enzyme resulted in 284.56 mg/g intracellular lipids, a 5.6-fold increase compared to the wild-type strain. This study provides some attractive strategies for increasing lipid production in E. coli by simulating the lipid accumulation of oleaginous microorganisms, which could aid the development of a prokaryotic fatty acid producer.

Ecological distribution of Spirillum lipoferum Beijerinck.

PubMed

Dobereiner, J; Marriel, I E; Nery, M

1976-10-01

A survey in various countries revealed that the N2-fixing Spirillum lipoferum Beijerinck is a very common root and soil inhabitant in the tropics. More than half of the grass root and soil samples collected in tropical countries (four African countries and Brazil) contained abundant S. lipoferum populations, while less than 10% of the samples collected in temperate South Brazil, Kenya, and the U.S.A. contained the organism. There is a pronounced vegetation effect. Panicum maximum seems the most favorable among the forage grasses, while few positive samples were found under virgin tropical forest. Legume roots contained less S. lipoferum than adjacent soils. More than 80% of the samples from cereal roots (maize, sorghum, wheat, and rye) grown in fields fertilized with PK and Mo, in Rio de Janeiro State, were positive. Maize and sorghum grown under similar conditions in Wisconsin contained less than 10% of positive samples, but when maize fields were inoculated 90% of the root samples contained S. lipoferum. Alluvial soils were more favorable than eroded hill soils. Occurrence in soil was strongly pH-dependent with a pH around 7, being optimal (correlation coefficient r = 0.90). Sporadic occurrence was observed even in soils with pH 4.8. Surface-sterilized P. maximum roots collected from soils with pH ranging from 4.8 to 7.2 contained high S. lipoferum numbers which did not correlate with soil pH (r = 0.41). Amendment with malate of acid soils was not very effective in increasing nitrogenase (N2-ase) activity, but in two soils with pH above 6.4, high N2-ase activity was obtained after 16 to 48 h of incubation. In two soils from a temperate climate region, inoculation with S. lipoferum increased N2-ase activity produced through malate amendment.

Relationships between the Efficiencies of Photosystems I and II and Stromal Redox State in CO2-Free Air 1

PubMed Central

Harbinson, Jeremy; Foyer, Christine H.

1991-01-01

The responses of the efficiencies of photosystems I and II, stromal redox state (as indicated by NADP-malate dehydrogenase activation state), and activation of the Benson-Calvin cycle enzymes ribulose 1,5-bisphosphate carboxylase and fructose 1,6-bisphosphatase to varying irradiance were measured in pea (Pisum sativum L.) leaves operating close to the CO2 compensation point. A comparison of the relationships among these parameters obtained from leaves in air was made with those obtained when the leaves were maintained in air from which the CO2 had been removed. P700 was more oxidized at any measured irradiance in CO2-free air than in air. The relationship between the quantum efficiencies of the photosystems in CO2-free air was distinctly curvilinear in contrast to the predominantly linear relationship obtained with leaves in air. This nonlinearity may be consistent with the operation of cyclic electron flow around photosystem I because the quantum efficiency of photosystem II was much more restricted than the quantum efficiency of photosystem I. In CO2-free air, measured NADP-malate dehydrogenase activities varied considerably at low irradiances. However, at high irradiance the activity of the enzyme was low, implying that the stroma was oxidized. In contrast, fructose-1,6-bisphosphatase activities tended to increase with increasing electron flux through the photosystems. Ribulose-1,5-bisphosphate carboxylase activity remained relatively constant with respect to irradiance in CO2-free air, with an activation state 50% of maximum. We conclude that, at the CO2 compensation point and high irradiance, low redox states are favored and that cyclic electron flow may be substantial. These two features may be the requirements necessary to trigger and maintain the dissipative processes in the thylakoid membrane. PMID:16668401

Impaired mitochondrial pyruvate importation in a patient and a fetus at risk.

PubMed

Brivet, M; Garcia-Cazorla, A; Lyonnet, S; Dumez, Y; Nassogne, M C; Slama, A; Boutron, A; Touati, G; Legrand, A; Saudubray, J M

2003-03-01

The patient was the first child of healthy consanguineous parents. She presented at birth with hypotonia, mild facial dysmorphism, periventricular cysts, marked metabolic acidosis, hyperlactacidemia with normal lactate/pyruvate molar ratios, normoglycemia, and normal ammonia. Hyperlactacidemia was severe (5-14 mmol/l) and not corrected with bicarbonate, thiamine (10 mg/d), 2-chloropropionate (100 mg/kg/d) and a ketogenic diet. Pyruvate dehydrogenase (PDHC) activity was normal in lymphocytes and fibroblasts. Functional assays were performed in digitonin-permeabilized fibroblasts to measure oxidation rates from radiolabeled pyruvate and malate. The production of [14C]acetylcarnitine or [14C]citric cycle intermediates derived from [2-14C]pyruvate as well as the release of 14CO(2) from [1-14C]pyruvate was severely impaired, whereas decarboxylation of [U-14C]malate was normal. With increasing concentrations of [1-14C]pyruvate, the patient's fibroblasts behave like control fibroblasts incubated in the presence of alpha-cyano-4-hydroxycinnamate, a specific inhibitor of mitochondrial pyruvate uptake: a progressive increase in 14CO(2) production was observed, likely due to passive diffusion of [1-14C]pyruvate through the mitochondrial membranes. Our results are consistent with a defect of mitochondrial pyruvate transport in the patient. Mutational analysis was precluded as the cDNA sequence of the pyruvate carrier has not been identified as yet in any organism. An affected fetus was recognized in a subsequent dichorionic twin pregnancy using the coupled assay measuring [2-14C]pyruvate oxidation rates on digitonin-permeabilized trophoblasts. After selective feticide, the pregnancy was uncomplicated with delivery at 37w of a healthy female, who is currently 2-month old. Copyright 2003 Elsevier Science (USA)

Citric Acid Metabolism in Resistant Hypertension: Underlying Mechanisms and Metabolic Prediction of Treatment Response.

PubMed

Martin-Lorenzo, Marta; Martinez, Paula J; Baldan-Martin, Montserrat; Ruiz-Hurtado, Gema; Prado, Jose Carlos; Segura, Julian; de la Cuesta, Fernando; Barderas, Maria G; Vivanco, Fernando; Ruilope, Luis Miguel; Alvarez-Llamas, Gloria

2017-11-01

Resistant hypertension (RH) affects 9% to 12% of hypertensive adults. Prolonged exposure to suboptimal blood pressure control results in end-organ damage and cardiovascular risk. Spironolactone is the most effective drug for treatment, but not all patients respond and side effects are not negligible. Little is known on the mechanisms responsible for RH. We aimed to identify metabolic alterations in urine. In addition, a potential capacity of metabolites to predict response to spironolactone was investigated. Urine was collected from 29 patients with RH and from a group of 13 subjects with pseudo-RH. For patients, samples were collected before and after spironolactone administration and were classified in responders (n=19) and nonresponders (n=10). Nuclear magnetic resonance was applied to identify altered metabolites and pathways. Metabolites were confirmed by liquid chromatography-mass spectrometry. Citric acid cycle was the pathway most significantly altered ( P <0.0001). Metabolic concentrations were quantified and ranged from ng/mL malate to μg/mL citrate. Citrate and oxaloacetate increased in RH versus pseudoresistant. Together with α-ketoglutarate and malate, they were able to discriminate between responders and nonresponders, being the 4 metabolites increased in nonresponders. Combined as a prediction panel, they showed receiver operating characteristiccurve with area under the curve of 0.96. We show that citric acid cycle and deregulation of reactive oxygen species homeostasis control continue its activation after hypertension was developed. A metabolic panel showing alteration before spironolactone treatment and predicting future response of patients is shown. These molecular indicators will contribute optimizing the rate of control of RH patients with spironolactone. © 2017 American Heart Association, Inc.

The response of carbon metabolism and antioxidant defenses of alfalfa nodules to drought stress and to the subsequent recovery of plants.

PubMed

Naya, Loreto; Ladrera, Ruben; Ramos, Javier; González, Esther M; Arrese-Igor, Cesar; Minchin, Frank R; Becana, Manuel

2007-06-01

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N(2)ase) activity. Exposure of plants to a moderate drought (leaf water potential of -1.3 MPa) had no effect on sucrose (Suc) synthase (SS) activity, but caused inhibition of N(2)ase activity (-43%), accumulation of succinate (+36%) and Suc (+58%), and up-regulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (-2.1 MPa) decreased N(2)ase (-82%) and SS (-30%) activities and increased malate (+40%), succinate (+68%), and Suc (+435%). There was also up-regulation (mRNA) of cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin expression, but decreased MoFe- and Fe-proteins. Rewatering of plants led to a partial recovery of the activity (75%) and proteins (>64%) of N(2)ase, a complete recovery of Suc, and a decrease of malate (-48%) relative to control. The increase in O(2) diffusion resistance, the decrease in N(2)ase-linked respiration and N(2)ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the up-regulation of antioxidant genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought conditions prior to any detectable effect on SS or leghemoglobin. We conclude that a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of N(2)ase activity in alfalfa nodules.

Inhibition of Krebs cycle and activation of glyoxylate cycle in the course of chronological aging of Saccharomyces cerevisiae. Compensatory role of succinate oxidation.

PubMed

Samokhvalov, V; Ignatov, V; Kondrashova, M

2004-01-01

We investigated oxidative processes in mitochondria of Saccharomyces cerevisiae grown on ethanol in the course of chronological aging. We elaborated a model of chronological aging that avoids the influence of exhaustion of medium, as well as the accumulation of toxic metabolites during aging. A decrease in total respiration of cells and, even more, of the contribution of respiration coupled with ATP-synthesis was observed during aging. Aging is also related with the decrease of the contribution of malonate-insensitive respiration. Activities of citrate-synthase (CS), alpha-ketoglutarate dehydrogenase (KGDH) and malate dehydrogenase (MDH) were threefold decreased. The activity of NADP-dependent isocitrate dehydrogenase (NADP-ICDH) decreased more significantly, while the activity of NAD-dependent isocitrate dehydrogenase (NAD-ICDH) fell even greater, being completely inactivated on the third week of aging. In contrast, succinate dehydrogenase (SDH), enzymes of glyoxylate cycle (GCL) (isocitrate lyase (ICL) and malate synthase (MLS)), and enzymes of ethanol oxidation (alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH)), were activated by 50% or more. The behavior of oxidative enzymes and metabolic pathways are apparently inherent to a more viable, long-lived cells in population, selected in the course of chronological aging. This selection allows cells to reveal the mechanism of their higher viability as caused by shunting of complete Krebs cycle by glyoxylate cycle, with a concomitant increased rate of the most efficient energy source, namely succinate formation and oxidation. Thiobarbituric-reactive species (TAR species) increased during aging. We supposed that to be the immediate cause of damage of a part of yeast population. These data show that a greater succinate contribution to respiration in more active cells is a general property of yeast and animal tissues.

Mitochondrial remodeling in the liver following chronic alcohol feeding to rats.

PubMed

Han, Derick; Johnson, Heather S; Rao, Madhuri P; Martin, Gary; Sancheti, Harsh; Silkwood, Kai H; Decker, Carl W; Nguyen, Kim Tho; Casian, Joseph G; Cadenas, Enrique; Kaplowitz, Neil

2017-01-01

The feeding of alcohol orally (Lieber-DeCarli diet) to rats has been shown to cause declines in mitochondrial respiration (state III), decreased expression of respiratory complexes, and decreased respiratory control ratios (RCR) in liver mitochondria. These declines and other mitochondrial alterations have led to the hypothesis that alcohol feeding causes "mitochondrial dysfunction" in the liver. If oral alcohol feeding leads to mitochondrial dysfunction, one would predict that increasing alcohol delivery by intragastric (IG) alcohol feeding to rats would cause greater declines in mitochondrial bioenergetics in the liver. In this study, we examined the mitochondrial alterations that occur in rats fed alcohol both orally and intragastrically. Oral alcohol feeding decreased glutamate/malate-, acetaldehyde- and succinate-driven state III respiration, RCR, and expression of respiratory complexes (I, III, IV, V) in liver mitochondria, in agreement with previous results. IG alcohol feeding, on the other hand, caused a slight increase in glutamate/malate-driven respiration, and significantly increased acetaldehyde-driven respiration in liver mitochondria. IG feeding also caused liver mitochondria to experience a decline in succinate-driven respiration, but these decreases were smaller than those observed with oral alcohol feeding. Surprisingly, oral and IG alcohol feeding to rats increased mitochondrial respiration using other substrates, including glycerol-3-phosphate (which delivers electrons from cytoplasmic NADH to mitochondria) and octanoate (a substrate for beta-oxidation). The enhancement of glycerol-3-phosphate- and octanoate-driven respiration suggests that liver mitochondria remodeled in response to alcohol feeding. In support of this notion, we observed that IG alcohol feeding also increased expression of mitochondrial glycerol phosphate dehydrogenase-2 (GPD2), transcription factor A (TFAM), and increased mitochondrial NAD + -NADH and NADP + -NADPH levels in the liver. Our findings suggest that mitochondrial dysfunction represents an incomplete picture of mitochondrial dynamics that occur in the liver following alcohol feeding. While alcohol feeding causes some mitochondrial dysfunction (i.e. succinate-driven respiration), our work suggests that the major consequence of alcohol feeding is mitochondrial remodeling in the liver as an adaptation. This mitochondrial remodeling may play an important role in the enhanced alcohol metabolism and other adaptations in the liver that develop with alcohol intake. Copyright © 2016 Elsevier Inc. All rights reserved.

Pears and renal stones: possible weapon for prevention? A comprehensive narrative review.

PubMed

Manfredini, R; De Giorgi, A; Storari, A; Fabbian, F

2016-01-01

Urinary stones have been recognized as a human disease since dawn of history and treatment of this condition is reported by Egyptian medical writings. Also, pears have a very long history, being one of the earliest cultivated fruit trees and also known for medicinal use. Urinary tract stone formation represents a common condition and also a significant burden for health care service, due also to possible frequent relapses. Furthermore, urinary stones have been reported to have relationship with different metabolic derangements, and appropriate diet could contribute to avoid or reduce urinary stone formation. Citrate is an inhibitor of crystal growth in the urinary system, and hypocitraturia represents a main therapeutical target in stone formers. Pears contain a significant amount of malic acid, a precursor of citrate, and have antioxidant activity as well. A diet supplemented with pears, and associated with low consumption of meat and salt could impact positively cardiometabolic risk and urinary tract stone formation. However, very few studies evaluated the impact of pears utilization on health, and none on urinary tract stone formation in particular. High content in malate could warrant protection against stone formation, avoiding patients at high risk to be compelled to assume a considerable and expensive amount of pills.

Assessing competence of broccoli consumption on inflammatory and antioxidant pathways in restraint-induced models: estimation in rat hippocampus and prefrontal cortex.

PubMed

Khalaj, Leila; Nejad, Sara Chavoshi; Mohammadi, Marzieh; Sarraf Zadeh, Sadaf; Pour, Marieh Hossein; Ashabi, Ghorbangol; Khodagholi, Fariba; Ahmadiani, Abolhassan

2013-01-01

A growing body of evidence advocated the protective and therapeutic potential of natural compounds and phytochemicals used in diets against pathological conditions. Herein, the outcome of dietary whole broccoli consumption prior to restraint stress has been investigated in the hippocampus and prefrontal cortex of male rats, two important regions involved in the processing of responses to stressful events. Interestingly, a region-specific effect was detected regarding some of antioxidant defense system factors: nuclear factor erythroid-derived 2-related factor 2 (Nrf-2) antioxidant pathway, mitochondrial prosurvival proteins involved in mitochondrial biogenesis, and apoptotic cell death proteins. Dietary broccoli supplementation modulated the restraint-induced changes towards a consistent overall protection in the hippocampus. In the prefrontal cortex, however, despite activation of most of the protective factors, presumably as an attempt to save the system against the stress insult, some detrimental outcomes such as induced malate dehydrogenase (MDA) level and cleaved form of caspase-3 were detectable. Such diversity may be attributed in one hand to the different basic levels and/or availability of defensive mechanisms within the two studied cerebral regions, and on the other hand to the probable dose-dependent and hormetic effects of whole broccoli. More experiments are essential to demonstrate these assumptions.

Localization, structure and polymorphism of two paralogous Xenopus laevis mitochondrial malate dehydrogenase genes.

PubMed

Tlapakova, Tereza; Krylov, Vladimir; Macha, Jaroslav

2005-01-01

Two paralogous mitochondrial malate dehydrogenase 2 (Mdh2) genes of Xenopus laevis have been cloned and sequenced, revealing 95% identity. Fluorescence in-situ hybridization (FISH) combined with tyramide amplification discriminates both genes; Mdh2a was localized into chromosome q3 and Mdh2b into chromosome q8. One kb cDNA probes detect both genes with 85% accuracy. The remaining signals were on the paralogous counterpart. Introns interrupt coding sequences at the same nucleotide as defined for mouse. Restriction polymorphism has been detected in the first intron of Mdh2a, while the individual variability in intron 6 of Mdh2b gene is represented by an insertion of incomplete retrotransposon L1Xl. Rates of nucleotide substitutions indicate that both genes are under similar evolutionary constraints. X. laevis Mdh2 genes can be used as markers for physical mapping and linkage analysis.

Anaplerotic roles of pyruvate carboxylase in mammalian tissues.

PubMed

Jitrapakdee, S; Vidal-Puig, A; Wallace, J C

2006-04-01

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC serves an anaplerotic role for the tricarboxylic acid cycle, when intermediates are removed for different biosynthetic purposes. In liver and kidney, PC provides oxaloacetate for gluconeogenesis. In adipocytes PC is involved in de novo fatty acid synthesis and glyceroneogenesis, and is regulated by the peroxisome proliferator-activated receptor-gamma, suggesting that PC is involved in the metabolic switch controlling fuel partitioning toward lipogenesis. In islets, PC is necessary for glucose-induced insulin secretion by providing oxaloacetate to form malate that participates in the 'pyruvate/malate cycle' to shuttle 3C or 4C between mitochondria and cytoplasm. Hyperglycemia and hyperlipidemia impair this cycle and affect glucose-stimulated insulin release. In astrocytes, PC is important for de novo synthesis of glutamate, an important excitatory neurotransmitter supplied to neurons. Transcriptional studies of the PC gene pinpoint some transcription factors that determine tissue-specific expression.

Neutron scattering reveals the dynamic basis of protein adaptation to extreme temperature.

PubMed

Tehei, Moeava; Madern, Dominique; Franzetti, Bruno; Zaccai, Giuseppe

2005-12-09

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 A) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.

Closing Plant Stomata Requires a Homolog of an Aluminum-Activated Malate Transporter

PubMed Central

Sasaki, Takayuki; Mori, Izumi C.; Furuichi, Takuya; Munemasa, Shintaro; Toyooka, Kiminori; Matsuoka, Ken; Murata, Yoshiyuki; Yamamoto, Yoko

2010-01-01

Plant stomata limit both carbon dioxide uptake and water loss; hence, stomatal aperture is carefully set as the environment fluctuates. Aperture area is known to be regulated in part by ion transport, but few of the transporters have been characterized. Here we report that AtALMT12 (At4g17970), a homolog of the aluminum-activated malate transporter (ALMT) of wheat, is expressed in guard cells of Arabidopsis thaliana. Loss-of-function mutations in AtALMT12 impair stomatal closure induced by ABA, calcium and darkness, but do not abolish either the rapidly activated or the slowly activated anion currents previously identified as being important for stomatal closure. Expressed in Xenopus oocytes, AtALMT12 facilitates chloride and nitrate currents, but not those of organic solutes. Therefore, we conclude that AtALMT12 is a novel class of anion transporter involved in stomatal closure. PMID:20154005

Closing plant stomata requires a homolog of an aluminum-activated malate transporter.

PubMed

Sasaki, Takayuki; Mori, Izumi C; Furuichi, Takuya; Munemasa, Shintaro; Toyooka, Kiminori; Matsuoka, Ken; Murata, Yoshiyuki; Yamamoto, Yoko

2010-03-01

Plant stomata limit both carbon dioxide uptake and water loss; hence, stomatal aperture is carefully set as the environment fluctuates. Aperture area is known to be regulated in part by ion transport, but few of the transporters have been characterized. Here we report that AtALMT12 (At4g17970), a homolog of the aluminum-activated malate transporter (ALMT) of wheat, is expressed in guard cells of Arabidopsis thaliana. Loss-of-function mutations in AtALMT12 impair stomatal closure induced by ABA, calcium and darkness, but do not abolish either the rapidly activated or the slowly activated anion currents previously identified as being important for stomatal closure. Expressed in Xenopus oocytes, AtALMT12 facilitates chloride and nitrate currents, but not those of organic solutes. Therefore, we conclude that AtALMT12 is a novel class of anion transporter involved in stomatal closure.

Nitrogen Metabolism in Plant Cell Suspension Cultures

PubMed Central

Behrend, Josef; Mateles, Richard I.

1976-01-01

Tobacco cells (Nicotiana tabacum) are capable of growth on ammonia as a sole nitrogen source only when succinate, malate, fumarate, citrate, α-ketoglutarate, glutamate, or pyruvate is added to the growth medium. A ratio between the molar concentrations of ammonia to succinate (as a complementary organic acid) in the growth medium of 1.5 was optimal. Succinate had no effect on the rate of uptake of ammonia from the medium into the cells although it did affect the intracellular concentration of ammonia. However, the changes were not sufficient to explain inhibition of growth as being due to ammonia toxicity. The radioactivity from 14C-succinate was incorporated into malate, glutamate, and aspartate within 2 minutes. It appears that the role of organic acids is neither connected to ammonium transport nor to relief of ammonia toxicity, but may be related to the need for additional carbon skeletons for synthesis of amino acids. PMID:16659706

EPR study of copper(II) ions in zinc 1-malate trihydrate

NASA Astrophysics Data System (ADS)

Bonomo, Raffaele P.; Di Bilio, Angel J.; Riggi, Francesco

1988-10-01

The EPR spectrum of Cu 2+ ions in zinc 1-malate trihydrate has been measured at 150 K for a large number of orientations of the applied magnetic field. Analysis yields the following spin Hamiltonian parameters: g x=2.0894±0.0009, A x=-12.0±1.5, g y=2.0879±0.0005, A y=-8.7±1.0, R=-0.7±1.5, g z=2.4249±0.0005, A z=-120.1±0.9, P=9.9±0.5, where the units of A and P are 10 4 cm -1. The Zeeman and hyperfine coupling tensors are coincident within 2°. The spectrum shows forbidden transitions with abnormal intensity due to a large quadrupolar interaction. The direction of g z points towards the hydroxyl oxygen while the g x and g y directions lie approximately along the metal-carboxylate oxygen bond.

Loss of Mitochondrial Malate Dehydrogenase Activity Alters Seed Metabolism Impairing Seed Maturation and Post-Germination Growth in Arabidopsis1[OPEN

PubMed Central

2016-01-01

Mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37) has multiple roles; the most commonly described is its catalysis of the interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. The roles of mMDH in Arabidopsis (Arabidopsis thaliana) seed development and germination were investigated in mMDH1 and mMDH2 double knockout plants. A significant proportion of mmdh1mmdh2 seeds were nonviable and developed only to torpedo-shaped embryos, indicative of arrested seed embryo growth during embryogenesis. The viable mmdh1mmdh2 seeds had an impaired maturation process that led to slow germination rates as well as retarded post-germination growth, shorter root length, and decreased root biomass. During seed development, mmdh1mmdh2 showed a paler green phenotype than the wild type and exhibited deficiencies in reserve accumulation and reduced final seed biomass. The respiration rate of mmdh1mmdh2 seeds was significantly elevated throughout their maturation, consistent with the previously reported higher respiration rate in mmdh1mmdh2 leaves. Mutant seeds showed a consistently higher content of free amino acids (branched-chain amino acids, alanine, serine, glycine, proline, and threonine), differences in sugar and sugar phosphate levels, and lower content of 2-oxoglutarate. Seed-aging assays showed that quiescent mmdh1mmdh2 seeds lost viability more than 3 times faster than wild-type seeds. Together, these data show the important role of mMDH in the earliest phases of the life cycle of Arabidopsis. PMID:27208265

D-malate production by permeabilized Pseudomonas pseudoalcaligenes; optimization of conversion and biocatalyst productivity.

PubMed

Michielsen, M J; Frielink, C; Wijffels, R H; Tramper, J; Beeftink, H H

2000-04-14

For the development of a continuous process for the production of solid D-malate from a Ca-maleate suspension by permeabilized Pseudomonas pseudoalcaligenes, it is important to understand the effect of appropriate process parameters on the stability and activity of the biocatalyst. Previously, we quantified the effect of product (D-malate2 -) concentration on both the first-order biocatalyst inactivation rate and on the biocatalytic conversion rate. The effects of the remaining process parameters (ionic strength, and substrate and Ca2 + concentration) on biocatalyst activity are reported here. At (common) ionic strengths below 2 M, biocatalyst activity was unaffected. At high substrate concentrations, inhibition occurred. Ca2+ concentration did not affect biocatalyst activity. The kinetic parameters (both for conversion and inactivation) were determined as a function of temperature by fitting the complete kinetic model, featuring substrate inhibition, competitive product inhibition and first-order irreversible biocatalyst inactivation, at different temperatures simultaneously through three extended data sets of substrate concentration versus time. Temperature affected both the conversion and inactivation parameters. The final model was used to calculate the substrate and biocatalyst costs per mmol of product in a continuous system with biocatalyst replenishment and biocatalyst recycling. Despite the effect of temperature on each kinetic parameter separately, the overall effect of temperature on the costs was found to be negligible (between 293 and 308 K). Within pertinent ranges, the sum of the substrate and biocatalyst costs per mmol of product was calculated to decrease with the influent substrate concentration and the residence time. The sum of the costs showed a minimum as a function of the influent biocatalyst concentration.

Plastidial NAD-Dependent Malate Dehydrogenase: A Moonlighting Protein Involved in Early Chloroplast Development Through its Interaction with an FtsH12-FtsHi Protease Complex.

PubMed

Schreier, Tina B; Antoine, Cléry; Schläfli, Michael; Galbier, Florian; Stadler, Martha; Demarsy, Emilie; Albertini, Daniele; Maier, Benjamin A; Kessler, Felix; Hörtensteiner, Stefan; Zeeman, Samuel C; Kötting, Oliver

2018-06-22

Malate dehydrogenases (MDH) convert malate to oxaloacetate using NAD(H) or NADP(H) as a cofactor. Arabidopsis thaliana mutants lacking plastidial NAD-dependent MDH (pdnad-mdh) are embryo-lethal, and constitutive silencing (miR-mdh-1) causes a pale, dwarfed phenotype. The reason for these severe phenotypes is unknown. Here, we rescued the embryo lethality of pdnad-mdh via embryo-specific expression of pdNAD-MDH. Rescued seedlings developed white leaves with aberrant chloroplasts and failed to reproduce. Inducible silencing of pdNAD-MDH at the rosette stage also resulted in white newly emerging leaves. These data suggest that pdNAD-MDH is important for early plastid development, which is consistent with the reductions in major plastidial galactolipid, carotenoid and protochlorophyllide levels in miR-mdh-1 seedlings. Surprisingly, the targeting of other NAD-dependent MDH isoforms to the plastid did not complement the embryo lethality of pdnad-mdh, while expression of enzymatically inactive pdNAD-MDH did. These complemented plants grew indistinguishably from the wild type. Both active and inactive forms of pdNAD-MDH interact with a heteromeric AAA-ATPase complex at the inner membrane of the chloroplast envelope. Silencing the expression of FtsH12, a key member of this complex, resulted in a phenotype that strongly resembles miR-mdh-1. We propose that pdNAD-MDH is essential for chloroplast development due to its moonlighting role in stabilizing FtsH12, distinct from its enzymatic function. © 2018 American Society of Plant Biologists. All rights reserved.

Plasticity of crassulacean acid metabolism at subtropical latitudes: a pineapple case study.

PubMed

Rainha, Nuno; Medeiros, Violante P; Câmara, Mariana; Faustino, Hélder; Leite, João P; Barreto, Maria do Carmo; Cruz, Cristina; Pacheco, Carlos A; Ponte, Duarte; Bernardes da Silva, Anabela

2016-01-01

Plants with the crassulacean acid metabolism (CAM) express high-metabolic plasticity, to adjust to environmental stresses. This article hypothesizes that irradiance and nocturnal temperatures are the major limitations for CAM at higher latitudes such as the Azores (37°45'N). Circadian CAM expression in Ananas comosus L. Merr. (pineapple) was assessed by the diurnal pattern of leaf carbon fixation into l-malate at the solstices and equinoxes, and confirmed by determining maximal phosphoenolpyruvate carboxylase (PEPC) activity in plant material. Metabolic adjustments to environmental conditions were confirmed by gas exchange measurements, and integrated with environmental data to determine CAM's limiting factors: light and temperature. CAM plasticity was observed at the equinoxes, under similar photoperiods, but different environmental conditions. In spring, CAM expression was similar between vegetative and flowering plants, while in autumn, flowering (before anthesis) and fructifying (with fully developed fruit before ripening) plants accumulated more l-malate. Below 100 µmol m(-2) s(-1) , CAM phase I was extended, reducing CAM phase III during the day. Carbon fixation inhibition may occur by two major pathways: nocturnal temperature (<15°C) inhibiting PEPC activity and l-malate accumulation; and low irradiance influencing the interplay between CAM phase I and III, affecting carboxylation and decarboxylation. Both have important consequences for plant development in autumn and winter. Observations were confirmed by flowering time prediction using environmental data, emphasizing that CAM expression had a strong seasonal regulation due to a complex network response to light and temperature, allowing pineapple to survive in environments not suitable for high productivity. © 2015 Scandinavian Plant Physiology Society.

The barley anion channel, HvALMT1, has multiple roles in guard cell physiology and grain metabolism.

PubMed

Xu, Muyun; Gruber, Benjamin D; Delhaize, Emmanuel; White, Rosemary G; James, Richard A; You, Jiangfeng; Yang, Zhenming; Ryan, Peter R

2015-01-01

The barley (Hordeum vulgare) gene HvALMT1 encodes an anion channel in guard cells and in certain root tissues indicating that it may perform multiple roles. The protein localizes to the plasma membrane and facilitates malate efflux from cells when constitutively expressed in barley plants and Xenopus oocytes. This study investigated the function of HvALMT1 further by identifying its tissue-specific expression and by generating and characterizing RNAi lines with reduced HvALMT1 expression. We show that transgenic plants with 18-30% of wild-type HvALMT1 expression had impaired guard cell function. They maintained higher stomatal conductance in low light intensity and lost water more rapidly from excised leaves than the null segregant control plants. Tissue-specific expression of HvALMT1 was investigated in developing grain and during germination using transgenic barley lines expressing the green fluorescent protein (GFP) with the HvALMT1 promoter. We found that HvALMT1 is expressed in the nucellar projection, the aleurone layer and the scutellum of developing barley grain. Malate release measured from isolated aleurone layers prepared from imbibed grain was significantly lower in the RNAi barley plants compared with control plants. These data provide molecular and physiological evidence that HvALMT1 functions in guard cells, in grain development and during germination. We propose that HvALMT1 releases malate and perhaps other anions from guard cells to promote stomatal closure. The likely roles of HvALMT1 during seed development and grain germination are also discussed. © 2014 Scandinavian Plant Physiology Society.

Interactive response of photosynthetic characteristics in Haloxylon ammodendron and Hedysarum scoparium exposed to soil water and air vapor pressure deficits.

PubMed

Gong, Chunmei; Wang, Jiajia; Hu, Congxia; Wang, Junhui; Ning, Pengbo; Bai, Juan

2015-08-01

C4 plants possess better drought tolerance than C3 plants. However, Hedysarum scoparium, a C3 species, is dominant and widely distributed in the desert areas of northwestern China due to its strong drought tolerance. This study compared it with Haloxylon ammodendron, a C4 species, regarding the interactive effects of drought stress and different leaf-air vapor pressure deficits. Variables of interest included gas exchange, the activity levels of key C4 photosynthetic enzymes, and cellular anatomy. In both species, gas exchange parameters were more sensitive to high vapor pressure deficit than to strong water stress, and the net CO2 assimilation rate (An) was enhanced as vapor pressure deficits increased. A close relationship between An and stomatal conductance (gs) suggested that the species shared a similar response mechanism. In H. ammodendron, the activity levels of key C4 enzymes were higher, including those of phosphoenolpyruvate carboxylase (PEPC) and nicotinamide adenine dinucleotide phosphate-malate enzyme (NADP-ME), whereas in H. scoparium, the activity level of nicotinamide adenine dinucleotide-malate enzyme (NAD-ME) was higher. Meanwhile, H. scoparium utilized adaptive structural features, including a larger relative vessel area and a shorter distance from vein to stomata, which facilitated the movement of water. These findings implied that some C4 biochemical pathways were present in H. scoparium to respond to environmental challenges. Copyright © 2015. Published by Elsevier B.V.

FrnE, a Cadmium-Inducible Protein in Deinococcus radiodurans, Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo

PubMed Central

Khairnar, Nivedita P.; Joe, Min-Ho; Misra, H. S.; Lim, Sang-Yong

2013-01-01

Deinococcus radiodurans R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including frnE (drfrnE) and some of those involved in DNA repair and oxidative stress tolerance. The drfrnE::nptII mutant of D. radiodurans showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ∼15- and ∼3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ∼2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In trans expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the dsbA mutant of Escherichia coli. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42°C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase in vitro and has a role in oxidative stress tolerance of D. radiodurans possibly by protecting the damaged cellular proteins from inactivation. PMID:23603741

Protection of rat skeletal muscle fibers by either L-carnitine or coenzyme Q10 against statins toxicity mediated by mitochondrial reactive oxygen generation

PubMed Central

La Guardia, P. G.; Alberici, L. C.; Ravagnani, F. G.; Catharino, R. R.; Vercesi, A. E.

2013-01-01

Mitochondrial redox imbalance has been implicated in mechanisms of aging, various degenerative diseases and drug-induced toxicity. Statins are safe and well-tolerated therapeutic drugs that occasionally induce myotoxicity such as myopathy and rhabdomyolysis. Previous studies indicate that myotoxicity caused by statins may be linked to impairment of mitochondrial functions. Here, we report that 1-h incubation of permeabilized rat soleus muscle fiber biopsies with increasing concentrations of simvastatin (1–40 μM) slowed the rates of ADP-or FCCP-stimulated respiration supported by glutamate/malate in a dose-dependent manner, but caused no changes in resting respiration rates. Simvastatin (1 μM) also inhibited the ADP-stimulated mitochondrial respiration supported by succinate by 24% but not by TMPD/ascorbate. Compatible with inhibition of respiration, 1 μM simvastatin stimulated lactate release from soleus muscle samples by 26%. Co-incubation of muscle samples with 1 mM L-carnitine, 100 μM mevalonate or 10 μM coenzyme Q10 (Co-Q10) abolished simvastatin effects on both mitochondrial glutamate/malate-supported respiration and lactate release. Simvastatin (1 μM) also caused a 2-fold increase in the rate of hydrogen peroxide generation and a decrease in Co-Q10 content by 44%. Mevalonate, Co-Q10 or L-carnitine protected against stimulation of hydrogen peroxide generation but only mevalonate prevented the decrease in Co-Q10 content. Thus, independently of Co-Q10 levels, L-carnitine prevented the toxic effects of simvastatin. This suggests that mitochondrial respiratory dysfunction induced by simvastatin, is associated with increased generation of superoxide, at the levels of complexes-I and II of the respiratory chain. In all cases the damage to these complexes, presumably at the level of 4Fe-4S clusters, is prevented by L-carnitine. PMID:23720630

Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G

PubMed Central

2011-01-01

Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues, and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H isoform, the catalytic mechanism is conserved. Sequence analysis in light of the structure indicates that additional members of isoform H likely exist in the databases but have been misannotated. PMID:21569248

Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

PubMed Central

Lodola, A; Shore, J D; Parker, D M; Holbrook, J

1978-01-01

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution. PMID:217361

An unbiased approach to identify genes involved in development in a turtle with temperature-dependent sex determination.

PubMed

Chojnowski, Jena L; Braun, Edward L

2012-07-15

Many reptiles exhibit temperature-dependent sex determination (TSD). The initial cue in TSD is incubation temperature, unlike genotypic sex determination (GSD) where it is determined by the presence of specific alleles (or genetic loci). We used patterns of gene expression to identify candidates for genes with a role in TSD and other developmental processes without making a priori assumptions about the identity of these genes (ortholog-based approach). We identified genes with sexually dimorphic mRNA accumulation during the temperature sensitive period of development in the Red-eared slider turtle (Trachemys scripta), a turtle with TSD. Genes with differential mRNA accumulation in response to estrogen (estradiol-17β; E(2)) exposure and developmental stages were also identified. Sequencing 767 clones from three suppression-subtractive hybridization libraries yielded a total of 581 unique sequences. Screening a macroarray with a subset of those sequences revealed a total of 26 genes that exhibited differential mRNA accumulation: 16 female biased and 10 male biased. Additional analyses revealed that C16ORF62 (an unknown gene) and MALAT1 (a long noncoding RNA) exhibited increased mRNA accumulation at the male producing temperature relative to the female producing temperature during embryonic sexual development. Finally, we identified four genes (C16ORF62, CCT3, MMP2, and NFIB) that exhibited a stage effect and five genes (C16ORF62, CCT3, MMP2, NFIB and NOTCH2) showed a response to E(2) exposure. Here we report a survey of genes identified using patterns of mRNA accumulation during embryonic development in a turtle with TSD. Many previous studies have focused on examining the turtle orthologs of genes involved in mammalian development. Although valuable, the limitations of this approach are exemplified by our identification of two genes (MALAT1 and C16ORF62) that are sexually dimorphic during embryonic development. MALAT1 is a noncoding RNA that has not been implicated in sexual differentiation in other vertebrates and C16ORF62 has an unknown function. Our results revealed genes that are candidates for having roles in turtle embryonic development, including TSD, and highlight the need to expand our search parameters beyond protein-coding genes.

Sunitinib Malate and Bevacizumab in Treating Patients With Kidney Cancer or Advanced Solid Malignancies

ClinicalTrials.gov

2014-04-01

Clear Cell Renal Cell Carcinoma; Recurrent Renal Cell Cancer; Stage I Renal Cell Cancer; Stage II Renal Cell Cancer; Stage III Renal Cell Cancer; Stage IV Renal Cell Cancer; Unspecified Adult Solid Tumor, Protocol Specific

Relations of enzymes inAspergillus repens grown under sodium chloride stress.

PubMed

Kelavkar, U P; Chhatpar, H S

1993-09-01

Aspergillus repens, a salt-pan isolate, was halotolerant. When grown for 72 h (log phase) and 144 h (beginning of stationary phase) in a medium containing 2M sodium chloride, the activities of invertase, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), and glutamate dehydrogenase (GDH) were found to have increased. Control cultures grown in a medium devoid of 2M NaCl failed to show such changes. The activities of MDH, G6PDH, and GDH increased with rising concentrations of Na(+) (as NaCl) when added up to 100MM in vitro. At higher concentrations they decreased. Changes in kinetic constants, Km and Vmax of these enzymes, as well as their de novo synthesis, were found to be some of the responses to NaCl stress-mediated changes.

Cabozantinib-s-malate in Treating Patients With Relapsed Osteosarcoma or Ewing Sarcoma

ClinicalTrials.gov

2018-05-23

Metastatic Ewing Sarcoma; Metastatic Osteosarcoma; Recurrent Ewing Sarcoma; Recurrent Osteosarcoma; Stage III Osteosarcoma AJCC v7; Stage IV Osteosarcoma AJCC v7; Stage IVA Osteosarcoma AJCC v7; Stage IVB Osteosarcoma AJCC v7; Unresectable Ewing Sarcoma; Unresectable Osteosarcoma

Effects of varying ruminally undegradable protein supplementation on forage digestion, nitrogen metabolism, and urea kinetics in Nellore cattle fed low-quality tropical forage.

PubMed

Batista, E D; Detmann, E; Titgemeyer, E C; Valadares Filho, S C; Valadares, R F D; Prates, L L; Rennó, L N; Paulino, M F

2016-01-01

Effects of supplemental RDP and RUP on nutrient digestion, N metabolism, urea kinetics, and muscle protein degradation were evaluated in Nellore heifers () consuming low-quality signal grass hay (5% CP and 80% NDF, DM basis). Five ruminally and abomasally cannulated Nellore heifers (248 ± 9 kg) were used in a 5 × 5 Latin square. Treatments were the control (no supplement) and RDP supplementation to meet 100% of the RDP requirement plus RUP provision to supply 0, 50, 100, or 150% of the RUP requirement. Supplemental RDP (casein plus NPN) was ruminally dosed twice daily, and RUP supply (casein) was continuously infused abomasally. Jugular infusion of [NN]-urea with measurement of enrichment in urine was used to evaluate urea kinetics. The ratio of urinary 3-methylhistidine to creatinine was used to estimate skeletal muscle protein degradation. Forage NDF intake (2.48 kg/d) was not affected ( ≥ 0.37) by supplementation, but supplementation did increase ruminal NDF digestion ( < 0.01). Total N intake (by design) and N retention increased ( < 0.001) with supplementation and also linearly increased with RUP provision. Urea entry rate and gastrointestinal entry rate of urea were increased by supplementation ( < 0.001). Supplementation with RUP linearly increased ( = 0.02) urea entry rate and tended ( = 0.07) to linearly increase gastrointestinal entry rate of urea. Urea use for anabolic purposes tended ( = 0.07) to be increased by supplementation, and RUP provision also tended ( = 0.08) to linearly increase the amount of urea used for anabolism. The fraction of recycled urea N incorporated into microbial N was greater ( < 0.001) for control (22%) than for supplemented (9%) heifers. Urinary 3-methylhistidine:creatinine of control heifers was more than double that of supplemented heifers ( < 0.001). Control heifers reabsorbed a greater ( < 0.001) fraction of urea from the renal tubule than did supplemented heifers. Overall, unsupplemented heifers had greater mobilization of AA from myofibrillar protein, which provided N for urea synthesis and subsequent recycling. Supplemental RUP, when RDP was supplied, not only increased N retention but also supported increased urea N recycling and increased ruminal microbial protein synthesis.

[Renal cell carcinoma with t(6;11)(p21.2;q13)/MALAT1-TFEB fusion: a clinical and pathological analysis].

PubMed

Xia, Qiuyuan; Shi, Shanshan; Shen, Qin; Wei, Xue; Wang, Xuan; Ma, Henghui; Lu, Zhenfeng; Zhou, Xiaojun; Rao, Qiu

2015-12-01

To study the clinicopathologic features, immunophenotype, differential diagnosis and prognosis of renal cell carcinoma (RCC) associated with t(6;11)(p21.2;q13)/MALAT1-TFEB gene fusion. A total of 9 cases of such rare tumor were selected for clinicopathologic, immunohistochemical and molecular analysis, with review of literature. The age of the patients ranged from 21 to 42 years (mean=31.3 years). The patients included four men and five women. Histologically, 4 of the 9 cases studied showed classic morphologic features of TFEB RCC, with hyaline material, pigments and psammoma bodies frequently identified. The remaining 5 cases demonstrated uncommon morphology, mimicking perivascular epithelioid cell neoplasm, clear cell RCC, chromophobe RCC or papillary RCC. Immunohistochemical study showed that TFEB and vimentin were positive in all cases. Most of the tumors studied also expressed Ksp-cadherin, E-cadherin, CD117, HMB45, Melan A and Cathepsin K. CKpan showed immunostaining in only 1 case. The staining for TFE3, CD10 and CK7 were all negative. TFEB gene rearrangement was detected in all the 9 cases studied using fluorescence in-situ hybridization. MALAT1-TFEB fusion gene was identified in 2 cases by polymerase chain reaction and direct sequencing. TFEB RCC seemed to be an indolent tumor. During a mean follow-up of 31 months, none developed tumor recurrence, progression, or metastasis. TFEB fusion-associated RCC is a rare neoplasm, tends to occur in young age group and carries an indolent behavior. Diagnosis relies on clinicopathologic findings and immunohistochemical analysis. TFEB break-apart FISH assay is a reliable tool in confirming the diagnosis.

Multiple polypeptides immunologically related to beta-poly(L-malate) hydrolase (polymalatase) in the plasmodium of the slime mold Physarum polycephalum.

PubMed

Karl, M; Holler, E

1998-01-15

Plasmodia of Physarum polycephalum contain large amounts of the cell-type-specific polyanion beta-poly(L-malate) and of a corresponding specific hydrolase (polymalatase), both expressed in the plasmodial form of the organism. We have partially purified polymalatase, the preparation consisting of several polypeptides, which could not be separated without destroying the hydrolase activity. Polypeptides of 68 kDa and 97 kDa were identified as polymalatases. Both were glycosylated, the 68-kDa form giving rise to a 54-kDa form when deglycosylated, and the 97-kDa form giving rise to an 88-kDa polypeptide that was indistinguishable from an 88-kDa inactive species also contained in the enzyme preparation. Antisera against each of these proteins were used to detect the intracellular distribution of the proteins. We found that the antisera crossreacted with the three proteins and, furthermore, with a multiplicity of polypeptides ubiquitously distributed over the plasmodium. Results of a two-dimensional non-denaturing in the first dimension and SDS-denaturing polyacrylamide gel electrophoresis in the second dimension suggested that the proteins were derived from a 200-kDa 'precursor' protein by proteolytic fragmentation. Polymalatase activity could be generated from a high molecular-mass precursor. According to several pieces of evidence, the proteolytic nicking occurred within plasmodia. The fragments were sticky and gave rise to preferred sizes of nicked macromolecules. The observed multiplicity varied as a function of the age of the cultures. The cellular distribution and the intracellular pH value were not compatible with an in situ polymalatase activity and suggested other, presently unknown, function(s) such as in the transportation of beta-poly(L-malate) from the nucleus to the culture medium.

Synthesis, growth, structural and optical studies of a new organic three dimensional framework: 4-(aminocarbonyl)pyridine 4-(aminocarbonyl)pyridinium hydrogen L-malate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vijayalakshmi, A.; Vidyavathy, B., E-mail: vidyavathybalraj@gmail.com; Peramaiyan, G.

2017-02-15

4-(aminocarbonyl)pyridine 4-(aminocarbonyl)pyridinium hydrogen L-malate [(4ACP)(4ACP).(LM)] a new organic nonlinear optical (NLO) crystal was grown by the slow evaporation method. Single crystal X-ray diffraction analysis revealed that the [(4ACP)(4ACP).(LM)] crystal belongs to monoclinic crystal system, space group P2{sub 1}/n, with a three dimensional network. Thermogravimetry (TG) and differential thermal (DT) analyses showed that [(4ACP)(4ACP).(LM)] is thermally stable up to 165 °C. The optical transmittance window and the lower cut-off wavelength of [(4ACP)(4ACP).(LM)] were found out by UV–vis–NIR spectral study. The molecular structure of [(4ACP)(4ACP).(LM)] was further confirmed by FTIR spectral studies. The relative dielectric permittivity and dielectric loss were determined asmore » function of frequency and temperature. The third order nonlinear optical property of [(4ACP)(4ACP).(LM)] was studied by the Z-scan technique using a 532 nm diode pumped CW Nd:YAG laser. Nonlinear refractive index, nonlinear absorption coefficient and third order nonlinear susceptibility of the grown crystal were found to be 7.38×10{sup −8} cm{sup 2}/W, 0.08×10{sup −4} cm/W and 5.36×10{sup −6} esu, respectively. The laser damage threshold value is found to be 1.75 GW/cm{sup 2} - Graphical abstract: In the crystal structure of the title complex, the asymmetric unit contains one hydrogen L-malate anion, 4-(aminocarbonyl)pyridinium cation and a neutral isonicotinamide molecule. It is stabilized by intermolecular N-H…O, C-H…O and O-H…O hydrogen bonds which generate a three dimensional network.« less

Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target.

PubMed

Hartuti, Endah Dwi; Inaoka, Daniel Ken; Komatsuya, Keisuke; Miyazaki, Yukiko; Miller, Russell J; Xinying, Wang; Sadikin, Mohamad; Prabandari, Erwahyuni Endang; Waluyo, Danang; Kuroda, Marie; Amalia, Eri; Matsuo, Yuichi; Nugroho, Nuki B; Saimoto, Hiroyuki; Pramisandi, Amila; Watanabe, Yoh-Ichi; Mori, Mihoko; Shiomi, Kazuro; Balogun, Emmanuel Oluwadare; Shiba, Tomoo; Harada, Shigeharu; Nozaki, Tomoyoshi; Kita, Kiyoshi

2018-03-01

Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc 1 complex inhibitor. Copyright © 2017 Elsevier B.V. All rights reserved.

Similar potential ATP-P production and enzymatic activities in the microplankton community off Concepción (Chile) under oxic and suboxic conditions

NASA Astrophysics Data System (ADS)

González, Rodrigo R.; Gutiérrez, Marcelo H.; Quiñones, Renato A.

2007-11-01

The effects of the oxygen minimum zone on the metabolism of the heterotrophic microplankton community (0.22-100 μm) in the Humboldt Current System, as well as the factors controlling its biomass production, remain unknown. Here we compare the effect of four sources of dissolved organic carbon (glucose, oxaloacetate, glycine, leucine) on microbial biomass production (such as ATP-P) and the potential enzymatic activities involved in catabolic pathways under oxic and suboxic conditions. Our results show significant differences ( p < 0.05) in the ATP-P production when induced by the different substrates that are used as dissolved organic carbon herein. The induction of ATP-P production is enhanced from glucose < oxaloacetate < glycine < leucine. Nevertheless, for individual substrates, no significant differences were found between incubation under oxic and suboxic conditions except in the case of leucine. For this amino acid, the induction of ATP-P synthesis was higher under suboxic than oxic conditions. The data sets of all the substrates used showed greater potential ATP-P production under suboxic than oxic conditions. The results of the potential enzymatic activities suggest that malate dehydrogenase has the highest signal of NADH oxidization activity in the microbial assemblage. Furthermore, for all experiments, the malate dehydrogenase activity data set had a significant relationship with ATP-P production. These findings suggest that the microbial community inhabiting the oxygen minimum zone has the same or greater potential growth than the community inhabiting more oxygenated strata of the water column and that malate dehydrogenase is the activity that best represents the metabolic potential of the community.

Chitosan Nanoencapsulated Exogenous Trypsin Biomimics Zymogen-Like Enzyme in Fish Gastrointestinal Tract

PubMed Central

Singh, Arvind R.; Ferosekhan, S.; Kothari, Dushyant C.; Pal, Asim Kumar; Jadhao, Sanjay Balkrishna

2013-01-01

Exogenous proteolytic enzyme supplementation is required in certain disease conditions in humans and animals and due to compelling reasons on use of more plant protein ingredients and profitability in animal feed industry. However, limitations on their utility in diet are imposed by their pH specificity, thermolabile nature, inhibition due to a variety of factors and the possibility of intestinal damage. For enhancing the efficacy and safety of exogenous trypsin, an efficient chitosan (0.04%) nanoencapsulation-based controlled delivery system was developed. An experiment was conducted for 45 days to evaluate nanoencapsulated trypsin (0.01% and 0.02%) along with 0.02% bare trypsin and 0.4% chitosan nanoparticles against a control diet on productive efficiency (growth rate, feed conversion and protein efficiency ratio), organo-somatic indices, nutrient digestibility, tissue enzyme activities, hematic parameters and intestinal histology of the fish Labeo rohita. All the synthesized nanoparticles were of desired characteristics. Enhanced fish productive efficiency using nanoencapsulated trypsin over its bare form was noticed, which corresponded with enhanced (P<0.01) nutrient digestibility, activity of intestinal protease, liver and muscle tissue transaminases (alanine and aspartate) and dehydrogenases (lactate and malate), serum blood urea nitrogen and serum protein profile. Intestinal tissues of fish fed with 0.02% bare trypsin showed broadened, marked foamy cells with lipid vacuoles. However, villi were healthier in appearance with improved morphological features in fish fed with nanoencapsulated trypsin than with bare trypsin, and the villi were longer in fish fed with 0.01% nanoencapsulated trypsin than with 0.02% nanoencapsulated trypsin. The result of this premier experiment shows that nanoencapsulated trypsin mimics zymogen-like proteolytic activity via controlled release, and hence the use of 0.01% nanoencapsulated trypsin (in chitosan nanoparticles) over bare trypsin can be favored as a dietary supplement in animals and humans. PMID:24040333

Central carbon metabolism in marine bacteria examined with a simplified assay for dehydrogenases.

PubMed

Wen, Weiwei; Wang, Shizhen; Zhou, Xiaofen; Fang, Baishan

2013-06-01

A simplified assay platform was developed to measure the activities of the key oxidoreductases in central carbon metabolism of various marine bacteria. Based on microplate assay, the platform was low-cost and simplified by unifying the reaction conditions of enzymes including temperature, buffers, and ionic strength. The central carbon metabolism of 16 marine bacteria, involving Pseudomonas, Exiguobacterium, Marinobacter, Citreicella, and Novosphingobium were studied. Six key oxidoreductases of central carbon metabolism, glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, 2-ketoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and isocitrate dehydrogenase were investigated by testing their activities in the pathway. High activity of malate dehydrogenase was found in Citreicella marina, and the specific activity achieved 22 U/mg in cell crude extract. The results also suggested that there was a considerable variability on key enzymes' activities of central carbon metabolism in some strains which have close evolutionary relationship while they adapted to the requirements of the niche they (try to) occupy.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation.

PubMed

Droux, M; Miginiac-Maslow, M; Jacquot, J P; Gadal, P; Crawford, N A; Kosower, N S; Buchanan, B B

1987-07-01

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Single step production of Cas9 mRNA for zygote injection.

PubMed

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

A family of highly conserved glycosomal 2-hydroxyacid dehydrogenases from Phytomonas sp.

PubMed

Uttaro, A D; Altabe, S G; Rider, M H; Michels, P A; Opperdoes, F R

2000-10-13

Phytomonas sp. contains two malate dehydrogenase isoforms, a mitochondrial isoenzyme with a high specificity for oxaloacetate and a glycosomal isozyme that acts on a broad range of substrates (Uttaro, A. D., and Opperdoes, F.R. (1997) Mol. Biochem. Parasitol. 89, 51-59). Here, we show that the low specificity of the latter isoenzyme is the result of a number of recent gene duplications that gave rise to a family of glycosomal 2-hydroxyacid dehydrogenase genes. Two of these genes were cloned, sequenced, and overexpressed in Escherichia coli. Although both gene products have 322 amino acids, share 90.4% identical residues, and have a similar hydrophobicity profile and net charge, their kinetic properties were strikingly different. One isoform behaved as a real malate dehydrogenase with a high specificity for oxaloacetate, whereas the other showed no activity with oxaloacetate but was able to reduce other oxoacids, such as phenyl pyruvate, 2-oxoisocaproate, 2-oxovalerate, 2-oxobutyrate, 2-oxo-4-methiolbutyrate, and pyruvate.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn,more » reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.« less

The viability of a nonenzymatic reductive citric acid cycle - Kinetics and thermochemistry

USGS Publications Warehouse

Ross, D.S.

2007-01-01

The likelihood of a functioning nonenzymatic reductive citric acid cycle, recently proposed as the precursor to biosynthesis on early Earth, is examined on the basis of the kinetics and thermochemistry of the acetate ??? pyruvate ??? oxaloacetate ??? malate sequence. Using data derived from studies of the Pd-catalyzed phosphinate reduction of carbonyl functions it is shown that the rate of conversion of pyruvate to malate with that system would have been much too slow to have played a role in the early chemistry of life, while naturally occurring reduction systems such as the fayalite-magnetite-quartz and pyrrhotite-pyrite-magnetite mineral assemblages would have provided even slower conversions. It is also shown that the production of pyruvate from acetate is too highly endoergic to be driven by a naturally occurring energy source such as pyrophosphate. It is thus highly doubtful that the cycle can operate at suitable rates without enzymes, and most unlikely that it could have participated in the chemistry leading to life. ?? 2006 Springer Science + Business Media B.V.

Alternative Pathway of Metronidazole Activation in Trichomonas vaginalis Hydrogenosomes

PubMed Central

Hrdý, Ivan; Cammack, Richard; Stopka, Pavel; Kulda, Jaroslav; Tachezy, Jan

2005-01-01

Metronidazole and related 5-nitroimidazoles are the only available drugs in the treatment of human urogenital trichomoniasis caused by the protozoan parasite Trichomonas vaginalis. The drugs are activated to cytotoxic anion radicals by their reduction within the hydrogenosomes. It has been established that electrons required for metronidazole activation are released from pyruvate by the activity of pyruvate:ferredoxin oxidoreductase and transferred to the drug by a low-redox-potential carrier, ferredoxin. Here we describe a novel pathway involved in the drug activation within the hydrogenosome. The source of electrons is malate, another major hydrogenosomal substrate, which is oxidatively decarboxylated to pyruvate and CO2 by NAD-dependent malic enzyme. The electrons released during this reaction are transferred from NADH to ferredoxin by NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I, which uses ferredoxin as electron acceptor. Trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from the hydrogenosomes. PMID:16304169

Localization of the enzymes involved in the photoevolution of H sub 2 from acetate in Chlamydomonas reinhardtii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willeford, K.O.; Gibbs, M.

1989-07-01

The localization of a series of enzymes involved in the anaerobic photodissimilation of acetate in Chlamydomonas reinhardtii F-60 adapted to a hydrogen metabolism was determined through the enzymatic analyses of the chloroplastic, cytoplasmic, and mitochondrial fractions obtained with a cellular fractionation procedure that incorporated cell wall removal by treatment with autolysine, digestion of the plasmalemma with the detergent digitonin, and fractionation by differential centrifugation on a Percoll step gradient. The sequence of events leading to the photoevolution of H{sub 2} from acetate includes the conversion of acetate into succinate via the extraplastidic glyoxylate cycle, the oxidation of succinate to fumaratemore » by chloroplastic succinate dehydrogenase, and the oxidation of malate to oxaloacetate in the chloroplast by NAD dependent malate dehydrogenase. The level of potential activity for the enzymes assayed were sufficient to accommodate the observed rate of the photoanaerobic dissimilation of acetate and the photoevolution of H{sub 2}.« less

The Viability of a Nonenzymatic Reductive Citric Acid Cycle Kinetics and Thermochemistry

NASA Astrophysics Data System (ADS)

Ross, David S.

2007-02-01

The likelihood of a functioning nonenzymatic reductive citric acid cycle, recently proposed as the precursor to biosynthesis on early Earth, is examined on the basis of the kinetics and thermochemistry of the acetate → pyruvate → oxaloacetate → malate sequence. Using data derived from studies of the Pd-catalyzed phosphinate reduction of carbonyl functions it is shown that the rate of conversion of pyruvate to malate with that system would have been much too slow to have played a role in the early chemistry of life, while naturally occurring reduction systems such as the fayalite magnetite quartz and pyrrhotite pyrite magnetite mineral assemblages would have provided even slower conversions. It is also shown that the production of pyruvate from acetate is too highly endoergic to be driven by a naturally occurring energy source such as pyrophosphate. It is thus highly doubtful that the cycle can operate at suitable rates without enzymes, and most unlikely that it could have participated in the chemistry leading to life.

Carbon and hydrogen metabolism of green algae in light and dark

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1990-01-01

After adaptation to a hydrogen metabolism, Chlamydomonas reinhardtii can photoanaerobically metabolize acetate with the evolution of H{sub 2} and CO{sub 2}. An enzyme profile of the chloroplastic, cytoplasmic, and mitochondrial fractions were obtained with a cellular fractionation procedure that incorporated cell wall removal by autolysine, digestion of the plasmalemma with digitonin and fractionation by differential centrifugation on a Percoll step gradient. The sequence of events leading to the photo-evolution of H{sub 2} from acetate includes the conversion of acetate into succinate via the extraplastidic glyoxylate cycle, the oxidation of succinate to fumarate by chloroplastic succinic dehydrogenase and the oxidation ofmore » malate to oxaloacetate in the chloroplast by NAD dependent malate dehydrogenase. The level of potential activity of the enzymes was sufficient to accommodate the observed rate of gas evolution. The isolated darkened chloroplast evolves aerobically CO{sub 2} from glucose indicating a chloroplastic respiratory pathway. Evolution of CO{sub 2} is blocked by mitochondrial inhibitors.« less

Metabolic behavior and enzymatic aspects of denitrifying EBPR sludge in a continuous-flow anaerobic-anoxic system.

PubMed

Zafiriadis, Ilias; Ntougias, Spyridon; Kapagiannidis, Anastasios G; Aivasidis, Alexander

2013-10-01

The metabolic aspects of enhanced biological phosphorus removal (EBPR) were investigated for the first time in a continuous-flow anaerobic-anoxic plant fed with acetate, propionate, or substrates which are involved in the tricarboxylic acid and/or glyoxylate cycle, i.e., fumarate, malate, or oxaloacetate, as the sole carbon source. Although the polyphosphate-accumulating organisms (PAOs) population remained stable with any carbon source examined, no typical EBPR metabolism was observed during fumarate, malate, or oxaloacetate utilization. Specific enzymatic activities related to EBPR were determined in activated sludge homogenates and directly correlated with the nutrient metabolic rates. The experimental results indicated the direct involvement of alkaline phosphatase, pyrophosphatase, and exopolyphosphatase in the denitrifying EBPR process. Metabolic aspects of glyoxylate cycle enzymes are discussed with regard to the biomass anaerobic and anoxic activity. Process performance was highly influenced by the kind of substrate utilized, indicating that specific metabolic pathways should be followed to favor efficient EBPR.

Stereochemistry and function of oxaloacetate keto-enol tautomerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Creighton, D.J.; Johnson, J.D.; Lambert, M.R.

1986-05-01

Oxaloacetate keto-enol tautomerase, partially purified from porcine kidney, catalyzes the conversions of enol- to keto-oxaloacetate by a mechanism in which solvent protons end up equally distributed between the two prochiral positions at C3 of keto-oxaloacetate. This conclusion is based upon the observation that when enzyme catalyzed ketonization is conducted in /sup 3/H/sub 2/O in the presence of excess malate dehydrogenase and NADH, only 50% of the /sup 3/H in the isolated (2S)-(3-/sup 3/H)malate is labilized to solvent upon treatment with fumarase. Either the tautomerase operates on the basis of a highly unusual stereomechanistic principle or tautomerase activity is not anmore » evolved property of the enzyme protein. As a result of an attempt to clarify the physiological importance of oxaloacetate tautomerase activity, keto-oxaloacetate was demonstrated to be directly transported across the inner membrane of rat liver mitochrondria, on the basis of the results of kinetic and isotope-trapping experiments.« less

Effects of fat and/or methionine hydroxy analog added to a molasses-urea-based supplement on ruminal and postruminal digestion and duodenal flow of nutrients in beef steers consuming low-quality lovegrass hay.

PubMed

Lopez, R; Pulsipher, G D; Guerra-Liera, J E; Soto-Navarro, S A; Balstad, L A; Petersen, M K; Dhuyvetter, D V; Brown, M S; Krehbiel, C R

2016-06-01

Five crossbred beef steers (initial BW = 338.6 ± 7.8 kg) fitted with ruminal and duodenal cannulas were used in a 5 × 5 Latin square design experiment to evaluate the effects of methionine hydroxy analog (MHA) and/or yellow grease (fat) added to a molasses-urea-based supplement on intake and characteristics of digestion. Steers were fed low-quality hay (long-stem lovegrass : 3.3% CP, 76.8% NDF; DM basis) ad libitum and supplemented with 0.91 kg/d (as fed) of 1 of 4 supplements in a 2 × 2 + 1 factorial arrangement of treatments. Supplemental treatments were 1) control (no supplement, NC); 2) molasses-urea liquid supplement (U); 3) U containing (as-fed basis) 1.65% MHA (UM); 4) U containing (as-fed basis) 12% fat (UF); and 5) U containing (as-fed basis) 1.65% MHA and 12% fat (UMF). Total and forage OM intake (kg/d and as % of BW) increased ( < 0.01) with molasses-urea, decreased ( ≤ 0.04) with MHA, and were not affected ( = 0.61) with fat supplementation. Total tract NDF digestibility increased ( = 0.01) with molasses-urea supplementation, and was less ( = 0.01) for fat than for nonfat supplementation. Total and microbial N flowing to the duodenum increased ( = 0.01) with molasses-urea supplementation. Although, total N flowing to duodenum was not affected ( = 0.27), microbial N decreased ( = 0.01), and nonammonia nonmicrobial N (NANMN) increased ( = 0.01) with fat supplementation. Extent of in situ OM and NDF digestibility at 96 h increased ( = 0.01) with molasses-urea supplementation, but were not affected ( ≥ 0.14) by either MHA or fat supplementation. Duodenal flow of total AA, essential AA, and nonessential AA increased ( ≤ 0.02) with molasses-urea supplementation. Total and nonessential serum AA concentration decreased ( < 0.01) with molasses-urea supplementation. Total ruminal VFA concentration increased ( = 0.01) with molasses-urea supplementation, and was not affected ( ≥ 0.14) by MHA or fat supplementation. Fat can be used in molasses-urea liquid supplements for cattle consuming low-quality forage to increase energy intake without negatively affecting forage intake or characteristics of digestion. However, adding MHA did not further improve the response to urea supplementation of cattle consuming low-quality forage. Conversely, the inclusion of MHA on urea supplement decreased forage intake.

Sex differences in the regulation of spatially distinct cardiac mitochondrial subpopulations.

PubMed

Ribeiro, Rogério Faustino; Ronconi, Karoline Sousa; Morra, Elis Aguiar; Do Val Lima, Patrícia Ribeiro; Porto, Marcella Leite; Vassallo, Dalton Valentim; Figueiredo, Suely Gomes; Stefanon, Ivanita

2016-08-01

Spatially distinct mitochondrial subpopulation may mediate myocardial pathology through permeability transition pore opening (MPTP). The goal of this study was to assess sex differences on the two spatially distinct mitochondrial subpopulations: subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM) based on morphology, membrane potential, mitochondrial function, oxidative phosphorylation, and MPTP. Aged matched Wistar rats were used to study SSM and IFM. Mitochondrial size was larger in SSM than in IFM in both genders. However, SSM internal complexity, yield, and membrane potential were higher in male than in female. The maximal rate of mitochondrial respiration, states 3 and 4, using glutamate + malate as substrate, were higher in IFM and SSM in the male group compared to female. The respiratory control ratio (RCR-state3/state 4), was not different in both SSM and IFM with glutamate + malate. The ADP:O ratio was found higher in IFM and SSM from female compared to males. When pyruvate was used, state 3 was found unchanged in both IFM and SSM, state 4 was also greater in male IFM compared to female. The RCR increased in the SSM while IFM remained the same. State 4 was higher in male SSM while in the IFM remained the same. The IFM presented a higher Ca(2+) retention capacity compared with SSM, however, there was a greater sensitivity to Ca(2+)-induced MPTP in SSM and IFM in the male group compared to female. In conclusion, our data show that spatially distinct mitochondrial subpopulations have sex-based differences in oxidative phosphorylation, morphology, and calcium retention capacity.

Effect of the Associated Methanogen Methanobrevibacter thaueri on the Dynamic Profile of End and Intermediate Metabolites of Anaerobic Fungus Piromyces sp. F1.

PubMed

Li, Yuanfei; Jin, Wei; Cheng, Yanfen; Zhu, Weiyun

2016-09-01

Although the scheme of metabolic pathways involved in the production of the major end products has been described, the dynamic profile of metabolites of anaerobic fungi co-cultured with methanogens is limited, especially for the intermediate metabolites. In the present study, the fermentation of the co-culture of Piromyces sp. F1 and Methanobrevibacter thaueri on glucose was investigated. The presence of methanogens shortened the growth lag time of anaerobic fungi and enhanced the total gas production. The occurrence of the maximum cell dry weight and the disappearance of most of the substrate were observed at 24 h for the co-culture and 48 h for the fungal mono-culture. In the co-culture, hydrogen was detected at a very low level during fermentation, and formate transitorily accumulated at 24 h and disappeared at 48 h, resulting in an increase of pH. Acetate was higher during the fermentation in the co-culture (P < 0.05), while lactate and ethanol were higher only in the initial stage of fermentation (P < 0.05). After 48 h, lactate in the mono-culture became much higher than that in the co-culture (P < 0.05), and ethanol tended to remain the same in both cultures. Moreover, malate tended to be exhausted in the co-culture, while it accumulated in the mono-culture. Citrate was also detected in both co-culture and mono-culture. Collectively, these results suggest that methanogen enhanced the malate pathway and weakened the lactate pathway of anaerobic fungus.

Antioxidant properties of sour cherries (Prunus cerasus L.): role of colorless phytochemicals from the methanolic extract of ripe fruits.

PubMed

Piccolella, Simona; Fiorentino, Antonio; Pacifico, Severina; D'Abrosca, Brigida; Uzzo, Piera; Monaco, Pietro

2008-03-26

Many edible plant metabolites are known to be useful as cellular antioxidants. In the search for antioxidative chemicals from native fruits of the Campania region of Italy, Prunus cerasus L., an acidic cherry widely used for culinary purposes, has been studied. Fruit crude extracts (MeOH, EtOAc, and hexane) were submitted to an antioxidative screening using specific assay media characterized from the presence of highly reactive radical species (DPPH*, ABTS*+, O2*-, NO) or lipoperoxidation markers. The reducing power of the samples was also determined. It was observed that the most polar extracts in MeOH and EtOAc were able to exercise a massive and dose-increasing antioxidative capacity. The peculiar efficacy of the same extracts was revealed by investigating their protein and deoxyribose oxidation capacity. A preliminary analysis of total phenol, flavonoid, and anthocyanin contents together with biological screening data put the basis on P. cerasus fruit phytochemical investigation of methanolic extract. Twenty secondary metabolites were isolated and characterized by spectroscopic (especially 1D and 2D NMR) and spectrometric techniques. 1-(4-Hydroxyphenyl)-1,2-ethanediol-1,2-bis-1-O-beta-D-glucopyranoside (3), (4-hydroxy-3-methoxyphenyl)methanol-1-O-beta-D-gentiobioside (4), epicatechin-3-malate (14), and epicatechin-3-(1''-methyl)malate (15) were isolated for the first time. All of the compounds were evaluated for their radical scavenging activity on DPPH*, O2*-, and NO. Flavonoids and quinic acid derivatives were found to be the more antioxidative substances.

Malate synthase gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Williams).

PubMed

Pua, Eng-Chong; Chandramouli, Sumana; Han, Ping; Liu, Pei

2003-01-01

Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl-CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS-1, from the banana fruit is reported. MaMS-1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS-1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS-1 also shared an extensive sequence homology (79-81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS-1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS-1 expression in FG fruit could be stimulated by treatment with 1 microl l(-1) exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene-inducible.

Relationships of Leaf Net Photosynthesis, Stomatal Conductance, and Mesophyll Conductance to Primary Metabolism: A Multispecies Meta-Analysis Approach1

PubMed Central

Gago, Jorge; Daloso, Danilo de Menezes; Flexas, Jaume; Fernie, Alisdair Robert

2016-01-01

Plant metabolism drives plant development and plant-environment responses, and data readouts from this cellular level could provide insights in the underlying molecular processes. Existing studies have already related key in vivo leaf gas-exchange parameters with structural traits and nutrient components across multiple species. However, insights in the relationships of leaf gas-exchange with leaf primary metabolism are still limited. We investigated these relationships through a multispecies meta-analysis approach based on data sets from 17 published studies describing net photosynthesis (A) and stomatal (gs) and mesophyll (gm) conductances, alongside the 53 data profiles from primary metabolism of 14 species grown in different experiments. Modeling results highlighted the conserved patterns between the different species. Consideration of species-specific effects increased the explanatory power of the models for some metabolites, including Glc-6-P, Fru-6-P, malate, fumarate, Xyl, and ribose. Significant relationships of A with sugars and phosphorylated intermediates were observed. While gs was related to sugars, organic acids, myo-inositol, and shikimate, gm showed a more complex pattern in comparison to the two other traits. Some metabolites, such as malate and Man, appeared in the models for both conductances, suggesting a metabolic coregulation between gs and gm. The resulting statistical models provide the first hints for coregulation patterns involving primary metabolism plus leaf water and carbon balances that are conserved across plant species, as well as species-specific trends that can be used to determine new biotechnological targets for crop improvement. PMID:26977088

Organ-specific metabolic responses to drought in Pinus pinaster Ait.

PubMed

de Miguel, Marina; Guevara, M Ángeles; Sánchez-Gómez, David; de María, Nuria; Díaz, Luis Manuel; Mancha, Jose A; Fernández de Simón, Brígida; Cadahía, Estrella; Desai, Nalini; Aranda, Ismael; Cervera, María-Teresa

2016-05-01

Drought is an important driver of plant survival, growth, and distribution. Water deficit affects different pathways of metabolism, depending on plant organ. While previous studies have mainly focused on the metabolic drought response of a single organ, analysis of metabolic differences between organs is essential to achieve an integrated understanding of the whole plant response. In this work, untargeted metabolic profiling was used to examine the response of roots, stems, adult and juvenile needles from Pinus pinaster Ait. full-sib individuals, subjected to a moderate and long lasting drought period. Cyclitols content showed a significant alteration, in response to drought in all organs examined, but other metabolites increased or decreased differentially depending on the analyzed organ. While a high number of flavonoids were only detected in aerial organs, an induction of the glutathione pathway was mainly detected in roots. This result may reflect different antioxidant mechanisms activated in aerial organs and roots. Metabolic changes were more remarkable in roots than in the other organs, highlighting its prominent role in the response to water stress. Significant changes in flavonoids and ascorbate metabolism were also observed between adult and juvenile needles, consistent with previously proven differential functional responses between the two developmental stages. Genetic polymorphisms in candidate genes coding for a Myb1 transcription factor and a malate dehydrogenase (EC 1.1.1.37) were associated with different concentration of phenylalanine, phenylpropanoids and malate, respectively. The results obtained will support further research on metabolites and genes potentially involved in functional mechanisms related to drought tolerance in trees. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Economic co-production of poly(malic acid) and pullulan from Jerusalem artichoke tuber by Aureobasidium pullulans HA-4D.

PubMed

Xia, Jun; Xu, Jiaxing; Liu, Xiaoyan; Xu, Jiming; Wang, Xingfeng; Li, Xiangqian

2017-02-23

poly(L-malic acid) (PMA) is a water-soluble polyester with many attractive properties in medicine and food industries, but the high cost of PMA fermentation has restricted its further application for large-scale production. To overcome this problem, PMA production from Jerusalem artichoke tubers was successfully performed. Additionally, a valuable exopolysaccharide, pullulan, was co-produced with PMA by Aureobasidum pullulans HA-4D. The Jerusalem artichoke medium for PMA and pullulan co-production contained only 100 g/L hydrolysate sugar, 30 g/L CaCO 3 and 1 g/L NaNO 3 . Compared with the glucose medium, the Jerusalem artichoke medium resulted in a higher PMA concentration (114.4 g/L) and a lower pullulan concentration (14.3 g/L) in a 5 L bioreactor. Meanwhile, the activity of pyruvate carboxylase and malate dehydrogenas was significantly increased, while the activity of α-phosphoglucose mutase, UDP-glucose pyrophosphorylase and glucosyltransferase was not affected. To assay the economic-feasibility, large-scale production in a 1 t fermentor was performed, yielding 117.5 g/L PMA and 15.2 g/L pullulan. In this study, an economical co-production system for PMA and pullulan from Jerusalem artichoke was developed. The medium for PMA and pullulan co-production was significantly simplified when Jerusalem artichoke tubers were used. With the simplified medium, PMA production was obviously stimulated, which would be associated with the improved activity of pyruvate carboxylase and malate dehydrogenas.

Structural flexibility and protein adaptation to temperature: Molecular dynamics analysis of malate dehydrogenases of marine molluscs.

PubMed

Dong, Yun-Wei; Liao, Ming-Ling; Meng, Xian-Liang; Somero, George N

2018-02-06

Orthologous proteins of species adapted to different temperatures exhibit differences in stability and function that are interpreted to reflect adaptive variation in structural "flexibility." However, quantifying flexibility and comparing flexibility across proteins has remained a challenge. To address this issue, we examined temperature effects on cytosolic malate dehydrogenase (cMDH) orthologs from differently thermally adapted congeners of five genera of marine molluscs whose field body temperatures span a range of ∼60 °C. We describe consistent patterns of convergent evolution in adaptation of function [temperature effects on K M of cofactor (NADH)] and structural stability (rate of heat denaturation of activity). To determine how these differences depend on flexibilities of overall structure and of regions known to be important in binding and catalysis, we performed molecular dynamics simulation (MDS) analyses. MDS analyses revealed a significant negative correlation between adaptation temperature and heat-induced increase of backbone atom movements [root mean square deviation (rmsd) of main-chain atoms]. Root mean square fluctuations (RMSFs) of movement by individual amino acid residues varied across the sequence in a qualitatively similar pattern among orthologs. Regions of sequence involved in ligand binding and catalysis-termed mobile regions 1 and 2 (MR1 and MR2), respectively-showed the largest values for RMSF. Heat-induced changes in RMSF values across the sequence and, importantly, in MR1 and MR2 were greatest in cold-adapted species. MDS methods are shown to provide powerful tools for examining adaptation of enzymes by providing a quantitative index of protein flexibility and identifying sequence regions where adaptive change in flexibility occurs.

Influence of supplementation with corn dried distillers grains plus solubles to growing calves fed medium-quality hay on growth performance and feeding behavior.

PubMed

Islas, A; Gilbery, T C; Goulart, R S; Dahlen, C R; Bauer, M L; Swanson, K C

2014-02-01

To determine the effect of increasing supplementation of corn dried distillers grains plus solubles (DDGS) on growth performance and feeding behavior, 70 steer calves (287 ± 10 kg of BW) were blocked by BW to 3 pens equipped with Insentec feeders. For 84 d, calves were fed medium-quality grass/legume hay offered for ad libitum intake and provided 1 of 3 dietary supplemental treatments (n = 7 or 8 steers per treatment within each pen; n = 23 or 24 per treatment): 1) nothing, 2) DDGS at 0.5% of BW daily (DM basis), and 3) DDGS at 1% of BW daily (DM basis). Hay intake (kg/d and % of BW daily) decreased linearly (P < 0.001) as DDGS supplementation increased. Total DMI (kg/d and % of BW) increased linearly (P < 0.001) with DDGS supplementation. Average daily gain and gain efficiency (G:F) responded quadratically (P ≤ 0.006) as G:F increased to a lesser extent when DDGS supplementation increased from 0.5 to 1% than from 0 to 0.5%. Meals (number per day) and time eating per meal for hay and total diet decreased linearly (P ≤ 0.006) with increasing DDGS supplementation. Time eating per day for hay responded quadratically (P < 0.001) and decreased to a greater extent when increasing from 0 to 0.5% DDGS supplementation than from 0.5 to 1% DDGS. Feed intake per minute (eating rate) for hay and total diet increased linearly (P ≤ 0.05) with increasing DDGS supplementation. On d 84, LM area, back fat thickness, and rump fat thickness increased linearly (P ≤ 0.006) with increasing DDGS supplementation. There were significant day × treatment interactions (P < 0.001) for plasma glucose and urea-N concentrations. Glucose did not change over the feeding period in control steers but increased in both supplemented groups. Urea-N decreased for control steers over the feeding period whereas urea-N increased in supplemented steers. In conclusion, supplementation of DDGS in amounts of 0.5 or 1% of BW daily can be used to reduce hay intake and improve ADG and G:F in growing steers fed medium-quality hay. Additionally, DDGS supplementation alters feeding behavior.

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.

PubMed Central

McCormack, J G

1985-01-01

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed. PMID:3000355

Sunitinib Malate in Treating Younger Patients With Recurrent, Refractory, or Progressive Malignant Glioma or Ependymoma

ClinicalTrials.gov

2015-08-18

Childhood Cerebellar Anaplastic Astrocytoma; Childhood Cerebral Anaplastic Astrocytoma; Childhood Cerebral Astrocytoma; Childhood Infratentorial Ependymoma; Childhood Mixed Glioma; Childhood Oligodendroglioma; Childhood Supratentorial Ependymoma; Recurrent Childhood Cerebellar Astrocytoma; Recurrent Childhood Cerebral Astrocytoma; Recurrent Childhood Ependymoma; Recurrent Childhood Subependymal Giant Cell Astrocytoma

Products of Dark CO2 Fixation in Pea Root Nodules Support Bacteroid Metabolism 1

PubMed Central

Rosendahl, Lis; Vance, Carroll P.; Pedersen, Walther B.

1990-01-01

Products of the nodule cytosol in vivo dark [14C]CO2 fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO2 fixation products was compared in effective (fix+) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix−) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The 14C incorporation from [14C]CO2 was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the 14C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining 14C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the 14C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive 14C labeling of malate and aspartate from nodule dark [14C]CO2 fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO2 fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more 14C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated 14C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained 14C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid 14C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of 14C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO2 fixation in the supply of the bacteroids with dicarboxylic acids. PMID:16667422

Daily supplementation with iron increases lipid peroxidation in young women with low iron stores.

PubMed

King, Sarah M; Donangelo, Carmen M; Knutson, Mitchell D; Walter, Patrick B; Ames, Bruce N; Viteri, Fernando E; King, Janet C

2008-06-01

The aim of this study was to determine whether women with low iron stores (plasma ferritin

Supplementation of Ascorbic Acid in Weanling Horses Following Prolonged Transportation

PubMed Central

Ralston, Sarah; Stives, Michelle

2012-01-01

Simple Summary Horses normally synthesize adequate amounts of ascorbic acid (vitamin C) in their liver to meet their needs for the vitamin. However, prolonged stress results in low plasma concentrations and reduced immune function. Weanling horses were supplemented with ascorbic acid for 5 or 10 days or no ascorbic acid (4 per group) following 50+ hours of transportation. Supplementation caused increases in plasma concentrations but both supplemented groups had decreased plasma ascorbic acid for 1 to 3 weeks following cessation of supplementation, possibly due to suppressed synthesis. Supplementation of ascorbic acid following prolonged stress will increase plasma concentrations, but prolonged supplementation should be avoided. Abstract Though horses synthesize ascorbic acid in their liver in amounts that meet their needs under normal circumstances, prolonged stress results in low plasma concentrations due to enhanced utilization and renal excretion and can reduce immune function. It was hypothesized that plasma ascorbic acid could be maintained in weanling horses by oral supplementation following prolonged transportation. Weanlings were supplemented with no ascorbic acid (Tx 0: n = 4), 5 grams ascorbic acid twice daily for 5 days (Tx 1: n = 4) or for 10 days (Tx 2: n = 4) following >50 hours of transportation. Supplementation caused slight (P < 0.2) increases in plasma ascorbic acid concentrations. Both supplemented groups had decreased (P < 0.05) plasma concentrations for 1 to 3 weeks following cessation of supplementation, possibly due to increased renal excretion or suppressed hepatic synthesis. Supplementation of ascorbic acid following prolonged stress will increase plasma concentrations, but prolonged supplementation should be avoided. PMID:26486916

Uncovering co-expression gene network regulating fruit acidity in diverse apples

USDA-ARS?s Scientific Manuscript database

Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that encodes an Aluminum-activated Malate Transporter1 (...

Nivolumab, Cabozantinib S-Malate, and Ipilimumab in Treating Patients With Recurrent Stage IV Non-small Cell Lung Cancer

ClinicalTrials.gov

2018-06-28

c-MET Gene Amplification; MET Exon 14 Mutation; Metastatic Non-Squamous Non-Small Cell Lung Carcinoma; Recurrent Non-Squamous Non-Small Cell Lung Carcinoma; RET/PTC Rearrangement; ROS1 Gene Rearrangement; Stage IV Non-Small Cell Lung Cancer AJCC v7

Capillarity, oxidative capacity and fibre composition of the soleus and gastrocnemius muscles of rats in hypothyroidism.

PubMed Central

Sillau, A H

1985-01-01

Muscle capillarity, mean and maximal diffusion distances and muscle fibre composition were evaluated in frozen sections stained for myosin ATPase of the soleus and the white area of the gastrocnemius medial head (gastrocnemius) of rats made hypothyroid by the injection of propylthiouracil (PTU) (50 mg kg-1) every day for 21 or 42 days. Oxygen consumption in the presence of excess ADP and Pi with pyruvate plus malate as substrates and the activity of cytochrome c oxidase were measured in muscle homogenates. Treatment with PTU decreased body oxygen consumption and the concentration of triiodothyronine in plasma. The capacity of the soleus and gastrocnemius muscles' homogenates to oxidize pyruvate plus malate and their cytochrome c oxidase activity were reduced after 21 or 42 days of treatment with PTU. Fibre composition in the soleus muscle was changed by treatment with PTU. There was a decrease in the proportion of type IIa or fast glycolytic oxidative fibres and an increase in type I or slow oxidative fibres. After 21 days of PTU administration there was also an increase in the proportion of fibres classified as IIc. The changes in fibre composition are believed to be the result of changes in the types of myosin synthesized by the fibres. Therefore, the fibres classified as IIc are, most probably, IIa fibres in the process of changing their myosin to that of the type I fibres. No changes in fibre composition were evident in the white area of the gastrocnemius medial head, an area made up of IIb or fast glycolytic fibres. The indices of capillarity: capillary density and capillary to fibre ratio, as well as mean and maximal diffusion distances from the capillaries, were not changed by the treatment with PTU in the muscles studied. The lack of changes in capillarity in spite of significant changes in oxidative capacity indicates that in skeletal muscle capillarity is not necessarily related to the oxidative capacity of the fibres. PMID:3989729

Relationships between the Efficiencies of Photosystems I and II and Stromal Redox State in CO(2)-Free Air : Evidence for Cyclic Electron Flow in Vivo.

PubMed

Harbinson, J; Foyer, C H

1991-09-01

The responses of the efficiencies of photosystems I and II, stromal redox state (as indicated by NADP-malate dehydrogenase activation state), and activation of the Benson-Calvin cycle enzymes ribulose 1,5-bisphosphate carboxylase and fructose 1,6-bisphosphatase to varying irradiance were measured in pea (Pisum sativum L.) leaves operating close to the CO(2) compensation point. A comparison of the relationships among these parameters obtained from leaves in air was made with those obtained when the leaves were maintained in air from which the CO(2) had been removed. P700 was more oxidized at any measured irradiance in CO(2)-free air than in air. The relationship between the quantum efficiencies of the photosystems in CO(2)-free air was distinctly curvilinear in contrast to the predominantly linear relationship obtained with leaves in air. This nonlinearity may be consistent with the operation of cyclic electron flow around photosystem I because the quantum efficiency of photosystem II was much more restricted than the quantum efficiency of photosystem I. In CO(2)-free air, measured NADP-malate dehydrogenase activities varied considerably at low irradiances. However, at high irradiance the activity of the enzyme was low, implying that the stroma was oxidized. In contrast, fructose-1,6-bisphosphatase activities tended to increase with increasing electron flux through the photosystems. Ribulose-1,5-bisphosphate carboxylase activity remained relatively constant with respect to irradiance in CO(2)-free air, with an activation state 50% of maximum. We conclude that, at the CO(2) compensation point and high irradiance, low redox states are favored and that cyclic electron flow may be substantial. These two features may be the requirements necessary to trigger and maintain the dissipative processes in the thylakoid membrane.

Diabetogenic milieus induce specific changes in mitochondrial transcriptome and differentiation of human pancreatic islets.

PubMed

Brun, Thierry; Li, Ning; Jourdain, Alexis A; Gaudet, Pascale; Duhamel, Dominique; Meyer, Jérémy; Bosco, Domenico; Maechler, Pierre

2015-09-15

In pancreatic β-cells, mitochondria play a central role in coupling glucose metabolism to insulin secretion. Chronic exposure of β-cells to metabolic stresses impairs their function and potentially induces apoptosis. Little is known on mitochondrial adaptation to metabolic stresses, i.e. high glucose, fatty acids or oxidative stress; being all highlighted in the pathogenesis of type 2 diabetes. Here, human islets were exposed for 3 days to 25 mm glucose, 0.4 mm palmitate, 0.4 mm oleate and transiently to H2O2. Culture at physiological 5.6 mm glucose served as no-stress control. Expression of mitochondrion-associated genes was quantified, including the transcriptome of mitochondrial inner membrane carriers. Targets of interest were further evaluated at the protein level. Three days after acute oxidative stress, no significant alteration in β-cell function or apoptosis was detected in human islets. Palmitate specifically increased expression of the pyruvate carriers MPC1 and MPC2, whereas the glutamate carrier GC1 and the aspartate/glutamate carrier AGC1 were down-regulated by palmitate and oleate, respectively. High glucose decreased mRNA levels of key transcription factors (HNF4A, IPF1, PPARA and TFAM) and energy-sensor SIRT1. High glucose also reduced expression of 11 mtDNA-encoded respiratory chain subunits. Interestingly, transcript levels of the carriers for aspartate/glutamate AGC2, malate DIC and malate/oxaloacetate/aspartate UCP2 were increased by high glucose, a profile suggesting important mitochondrial anaplerotic/cataplerotic activities and NADPH-generating shuttles. Chronic exposure to high glucose impaired glucose-stimulated insulin secretion, decreased insulin content, promoted caspase-3 cleavage and cell death, revealing glucotoxicity. Overall, expression profile of mitochondrion-associated genes was selectively modified by glucose, delineating a glucotoxic-specific signature. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Supplemental Citrulline Is More Efficient Than Arginine in Increasing Systemic Arginine Availability in Mice.

PubMed

Agarwal, Umang; Didelija, Inka C; Yuan, Yang; Wang, Xiaoying; Marini, Juan C

2017-04-01

Background: Arginine is considered to be an essential amino acid in various (patho)physiologic conditions of high demand. However, dietary arginine supplementation suffers from various drawbacks, including extensive first-pass extraction. Citrulline supplementation may be a better alternative than arginine, because its only fate in vivo is conversion into arginine. Objective: The goal of the present research was to determine the relative efficiency of arginine and citrulline supplementation to improve arginine availability. Methods: Six-week-old C57BL/6J male mice fitted with gastric catheters were adapted to 1 of 7 experimental diets for 2 wk. The basal diet contained 2.5 g l-arginine/kg, whereas the supplemented diets contained an additional 2.5, 7.5, and 12.5 g/kg diet of either l-arginine or l-citrulline. On the final day, after a 3-h food deprivation, mice were continuously infused intragastrically with an elemental diet similar to the dietary treatment, along with l-[ 13 C 6 ]arginine, to determine the splanchnic first-pass metabolism (FPM) of arginine. In addition, tracers were continuously infused intravenously to determine the fluxes and interconversions between citrulline and arginine. Linear regression slopes were compared to determine the relative efficiency of each supplement. Results: Whereas all the supplemented citrulline (105% ± 7% SEM) appeared in plasma and resulted in a marginal increase of 86% in arginine flux, supplemental arginine underwent an ∼70% FPM, indicating that only 30% of the supplemental arginine entered the peripheral circulation. However, supplemental arginine did not increase arginine flux. Both supplements linearly increased ( P < 0.01) plasma arginine concentration from 109 μmol/L for the basal diet to 159 and 214 μmol/L for the highest arginine and citrulline supplementation levels, respectively. However, supplemental citrulline increased arginine concentrations to a greater extent (35%, P < 0.01). Conclusions: Citrulline supplementation is more efficient at increasing arginine availability than is arginine supplementation itself in mice. © 2017 American Society for Nutrition.

Supplemental Citrulline Is More Efficient Than Arginine in Increasing Systemic Arginine Availability in Mice123

PubMed Central

Agarwal, Umang; Didelija, Inka C; Yuan, Yang; Wang, Xiaoying; Marini, Juan C

2017-01-01

Background: Arginine is considered to be an essential amino acid in various (patho)physiologic conditions of high demand. However, dietary arginine supplementation suffers from various drawbacks, including extensive first-pass extraction. Citrulline supplementation may be a better alternative than arginine, because its only fate in vivo is conversion into arginine. Objective: The goal of the present research was to determine the relative efficiency of arginine and citrulline supplementation to improve arginine availability. Methods: Six-week-old C57BL/6J male mice fitted with gastric catheters were adapted to 1 of 7 experimental diets for 2 wk. The basal diet contained 2.5 g l-arginine/kg, whereas the supplemented diets contained an additional 2.5, 7.5, and 12.5 g/kg diet of either l-arginine or l-citrulline. On the final day, after a 3-h food deprivation, mice were continuously infused intragastrically with an elemental diet similar to the dietary treatment, along with l-[13C6]arginine, to determine the splanchnic first-pass metabolism (FPM) of arginine. In addition, tracers were continuously infused intravenously to determine the fluxes and interconversions between citrulline and arginine. Linear regression slopes were compared to determine the relative efficiency of each supplement. Results: Whereas all the supplemented citrulline (105% ± 7% SEM) appeared in plasma and resulted in a marginal increase of 86% in arginine flux, supplemental arginine underwent an ∼70% FPM, indicating that only 30% of the supplemental arginine entered the peripheral circulation. However, supplemental arginine did not increase arginine flux. Both supplements linearly increased (P < 0.01) plasma arginine concentration from 109 μmol/L for the basal diet to 159 and 214 μmol/L for the highest arginine and citrulline supplementation levels, respectively. However, supplemental citrulline increased arginine concentrations to a greater extent (35%, P < 0.01). Conclusions: Citrulline supplementation is more efficient at increasing arginine availability than is arginine supplementation itself in mice. PMID:28179487

Fortified tuna bone powder supplementation increases bone mineral density of lactating rats and their offspring.

PubMed

Suntornsaratoon, Panan; Charoenphandhu, Narattaphol; Krishnamra, Nateetip

2018-03-01

Breastfeeding leads to bone calcium loss for milk production, resulting in progressive maternal osteopenia. Calcium supplement from natural sources has been postulated to be more beneficial to bone health than purified CaCO 3 because natural sources also contain other nutrients such as certain amino acids that might enhance calcium metabolism. Herein, we examined the effect of calcium supplementation from tuna bone powder and CaCO 3 on bones of dams and the offspring. Both forms of calcium supplement, i.e. tuna bone powder and CaCO 3 , increased maternal bone mineral density (BMD). However, bone histomorphometry revealed that only tuna bone had beneficial effect on maternal bone microstructure, i.e. increased bone formation, decreased bone resorption and increased in bone volume. Regarding the mechanical properties, the decreased ultimate load in non-supplement lactating mothers was restored to the load seen in nulliparous animals by calcium supplementation. Moreover, both tuna bone and CaCO 3 supplementation in mothers led to increased milk calcium concentration and consequently increased BMD in the growing offspring. Calcium supplement from tuna bone powder was effective in preventing maternal osteopenia. Tuna bone, which is a readily available fishing industrial waste, is a good alternative source of calcium supplement that increases BMD in both lactating mothers and the neonates. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Muscle Mass and Weight Gain Nutritional Supplements

NASA Astrophysics Data System (ADS)

Campbell, Bill

There are numerous sports supplements available that claim to increase lean body mass. However, for these sports supplements to exert any favorable changes in lean body mass, they must influence those factors regulating skeletal muscle hypertrophy (i.e., satellite cell activity, gene transcription, protein translation). If a given sports supplement does favorably influence one of these regulatory factors, the result is a positive net protein balance (in which protein synthesis exceeds protein breakdown). Sports supplement categories aimed at eliciting a positive net protein balance include anabolic hormone enhancers, nutrient timing pre- and postexercise workout supplements, anticatabolic supplements, and nitric oxide boosters. Of all the sports supplements available, only a few have been subject to multiple clinical trials with repeated favorable outcomes relative to increasing lean body mass. This chapter focuses on these supplements and others that have a sound theoretical rationale in relation to increasing lean body mass.

Effects of concentrate-to-forage ratios and 2-methylbutyrate supplementation on ruminal fermentation, bacteria abundance and urinary excretion of purine derivatives in Chinese Simmental steers.

PubMed

Wang, C; Liu, Q; Guo, G; Huo, W J; Pei, C X; Zhang, S L; Wang, H

2018-05-01

This study evaluated the effects of dietary concentrate levels and 2-methylbutyrate (2MB) supplementation on performance, ruminal fermentation, bacteria abundance, microbial enzyme activity and urinary excretion of purine derivatives (PD) in steers. Eight ruminally cannulated Simmental steers (12 months of age; 389 ± 3.7 kg of body weight) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement. Moderate-concentrate (400 g/kg diet [MC]) or high-concentrate (600 g/kg diet [HC]) diets were fed with or without 2MB (0 g/day [2MB-] or 15.0 g/day [2MB+]). Dry matter intake and average daily gain increased, but feed conversion ratio decreased with the HC diet or 2MB supplementation. Ruminal pH decreased, but total volatile fatty acid increased with the HC diet or 2MB supplementation. Molar proportion of acetate and acetate-to-propionate ratio decreased with the HC diet, but increased with 2MB supplementation. Propionate molar proportion and ruminal NH 3 -N content increased with the HC diet, but decreased with 2MB supplementation. Neutral detergent fibre degradability decreased with the HC diet, but increased with 2MB supplementation. Crude protein degradability increased with the HC diet or 2MB supplementation. Abundance of Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes and Bufyrivibrio fibrisolvens as well as activities of carboxymethyl cellulase, cellobiase, xylanase and pectinase decreased with the HC diet, but increased with 2MB supplementation. However, abundance of Prevotella ruminicola and Ruminobacter amylophilus as well as activities of α-amylase and protease increased with the HC diet or 2MB supplementation. Total PD excretion also increased with the HC diet or 2MB supplementation. The results suggested that growth performance, ruminal fermentation, CP degradability and total PD excretion increased with increasing dietary concentrate level from 40% to 60% or 2MB supplementation. The observed diet × 2MB interaction indicated that supplementation of 2MB was more efficacious for improving growth performance, ruminal fermentation and total PD excretion with promoted ruminal bacteria abundance and enzyme activity in the MC diet than in the HC diet. © 2018 Blackwell Verlag GmbH.

Effects of different levels of supplementation of a 50:50 mixture of molasses:crude glycerol on performance, Bermuda grass hay intake, and nutrient digestibility of beef cattle.

PubMed

Ciriaco, F M; Henry, D D; Mercadante, V R G; Schulmeister, T; Ruiz-Moreno, M; Lamb, G C; DiLorenzo, N

2015-05-01

Two experiments were performed to evaluate the effects of different levels of supplementation with a 50:50 (as-fed) mixture of molasses:crude glycerol on animal performance, total tract digestibility of nutrients, and ruminal in situ degradability of nutrients in beef heifers and steers consuming Tifton 85 Bermuda grass (Cynodon spp.) hay. For Exp. 1, 24 Angus crossbred heifers (380 ± 31 kg BW) were used in a generalized randomized block design. For Exp. 2, 8 ruminally cannulated Angus crossbred steers (323 ± 42 kg BW) were used in a 4 × 4 duplicated Latin square design. For both experiments, animals were housed in individual pens at the University of Florida Feed Efficiency Facility, had ad libitum access to Tifton 85 Bermuda grass hay, and were randomly assigned to 1 of 4 treatments: 1) CTRL, no supplementation; 2) SUP1, 0.45 kg/d (as fed) of 50:50 mixture; 3) SUP3, 1.36 kg/d (as fed) of 50:50 mixture; and 4) SUP5, 2.27 kg/d (as fed) of a 50:50 mixture. Individual feed intake was recorded. Total DMI increased linearly (P = 0.005) as the level of supplementation increased. Hay intake ranged from 1.36 (CTRL) to 1.23% (SUP5) of BW, and was not affected (P ≥ 0.10) by liquid supplementation. Final BW was not affected by liquid supplementation ( ≥ 0.10). There was a linear increase (P = 0.027) in ADG as the liquid supplementation amounts increased. Liquid supplementation did not affect G:F (P ≥ 0.10). Apparent total tract digestibility of DM, OM, NDF, and ADF increased linearly (P < 0.001), while CP total tract digestibility decreased linearly (P = 0.002) as the level of supplementation increased. Ruminal pH was decreased linearly (P = 0.012) as the level of supplementation increased. No effect (P ≥ 0.10) of liquid supplementation was detected on lag time for NDF and ADF content of bermudagrass hay; however, rate of degradation (Kd) of NDF tended (P = 0.076) to be affected cubically by liquid supplementation. In addition, liquid supplementation linearly decreased (P < 0.05) ED of OM, CP, NDF, and ADF. In conclusion, supplementing up to 2.27 kg/d of a 50:50 mixture of molasses:crude glycerol may stimulate microbial growth and fermentative activity, thereby increasing nutrient digestibility. Increased fiber digestion, along with energy supplementation, led to increased ADG in heifers consuming Bermuda grass hay.

Partial prevention of long-term femoral bone loss in aged ovariectomized rats supplemented with choline-stabilized orthosilicic acid.

PubMed

Calomme, M; Geusens, P; Demeester, N; Behets, G J; D'Haese, P; Sindambiwe, J B; Van Hoof, V; Vanden Berghe, D

2006-04-01

Silicon (Si) deficiency in animals results in bone defects. Choline-stabilized orthosilicic acid (ch-OSA) was found to have a high bioavailability compared to other Si supplements. The effect of ch-OSA supplementation was investigated on bone loss in aged ovariectomized (OVX) rats. Female Wistar rats (n = 58, age 9 months) were randomized in three groups. One group was sham-operated (sham, n = 21), and bilateral OVX was performed in the other two groups. OVX rats were supplemented orally with ch-OSA over 30 weeks (OVX1, n = 20; 1 mg Si/kg body weight daily) or used as controls (OVX0, n = 17). The serum Si concentration and the 24-hour urinary Si excretion of supplemented OVX rats was significantly higher compared to sham and OVX controls. Supplementation with ch-OSA significantly but partially reversed the decrease in Ca excretion, which was observed after OVX. The increase in bone turnover in OVX rats tended to be reduced by ch-OSA supplementation. ch-OSA supplementation increased significantly the femoral bone mineral content (BMC) in the distal region and total femoral BMC in OVX rats, whereas lumbar BMC was marginally increased. Femoral BMD was significantly increased at two sites in the distal region in OVX rats supplemented with ch-OSA compared to OVX controls. Total lumbar bone mineral density was marginally increased by ch-OSA supplementation. In conclusion, ch-OSA supplementation partially prevents femoral bone loss in the aged OVX rat model.

Intake, digestibility, and rumen and metabolic characteristics of cattle fed low-quality tropical forage and supplemented with nitrogen and different levels of starch.

PubMed

de Oliveira Franco, Marcia; Detmann, Edenio; de Campos Valadares Filho, Sebastião; Batista, Erick Darlisson; de Almeida Rufino, Luana Marta; Barbosa, Marcília Medrado; Lopes, Alexandre Ribeiro

2017-06-01

Effects of nitrogen supplementation associated with different levels of starch on voluntary intake, digestibility, and rumen and metabolic characteristics of cattle fed low-quality tropical forage ( Brachiaria decumbens hay, 7.4% crude protein, CP) were evaluated using ruminal and abomasal cannulated steers. Five European×Zebu young bulls (186 kg body weight, BW) were distributed according to a 5×5 Latin square. The following treatments were evaluated: control, supplementation with 300 g CP/d (0:1), supplementation with 300 g starch/d and 300 g CP/d (1:1), supplementation with 600 g starch/d and 300 g CP/d (2:1), and supplementation with 900 g starch/d and 300 g CP/d (3:1). A mixture of nitrogenous compounds provided 1/3 from true protein (casein) and 2/3 from non-protein nitrogen (mixture of urea and ammonium sulphate, 9:1) was used as the nitrogen supplement. In order to supply energy a unique source of corn starch was used. Supplements increased (p<0.05) dry matter intake, but did not affect (p>0.05) forage intake. There was a cubic effect (p<0.05) of starch on voluntary intake. This was attributed to the highest forage intake (g/kg BW) when using the 2:1 starch:CP ratio. Supplements increased (p<0.05) organic matter (OM) digestibility, but did not affect (p>0.05) neutral detergent fibre corrected for ash and protein (NDFap) digestibility. There was a positive linear effect (p<0.05) of the amount of starch supplemented on OM digestibility. Total NDFap digestibility was not affected (p>0.05) by the amount of supplemental starch. Ruminal ammonia nitrogen concentrations were higher (p<0.05) in supplemented animals, however, a negative linear effect (p<0.05) of amount of starch was observed. Supplements increased (p<0.05) the nitrogen balance (NB) and efficiency of nitrogen utilization. These effects were attributed to increased body anabolism, supported by higher (p<0.05) serum concentration of insulin-like growth factor 1. Increasing the amount of starch tended (p<0.06) to linearly increase the NB. In spite of this, there was a highest NB value for the 2:1 starch:CP ratio amongst the treatments with supplementation. Nitrogen supplementation in cattle fed low-quality tropical forage increases nitrogen retention in the animal's body. An additional supply of starch increases nitrogen retention by increasing energy availability for both rumen and animal metabolism.

Effect of Supplemental Corn Dried Distillers Grains with Solubles Fed to Beef Steers Grazing Native Rangeland during the Forage Dormant Season

PubMed Central

Murillo, M.; Herrera, E.; Ruiz, O.; Reyes, O.; Carrete, F. O.; Gutierrez, H.

2016-01-01

Two experiments were conducted to evaluate the effects of the level of corn dry distillers grains with solubles (CDDGS) supplementation on growing performance, blood metabolites, digestion characteristics and ruminal fermentation patterns in steers grazing dormant forage. In Exp. 1, of growth performance, 120 steers (204±5 kg initial body weight [BW]) were distributed randomly into 3 groups (each of 40 steers), which were provided with the following levels of CDDGS supplement: 0%, 0.25%, or 0.50% BW. All groups of steers were grazed for 30 days in each of 3 grazing periods (March, April, and May). Approximately 1,000 ha of the land was divided with electric fencing into 3 equally sized pastures (333 ha in size). Blood samples were collected monthly from 20 steers in each grazing group for analysis of glucose (G), urea-nitrogen (UN) and non-esterified fatty acids. Final BW, average daily gain (ADG) and supplement conversion (CDDGS-C) increased with increasing levels of CDDGS supplementation (p<0.05).The CDDGS supplementation also increased the plasma G and UN concentrations (p<0.05). In Exp. 2, of digestive metabolism, 9 ruminally cannulated steers (BW = 350±3 kg) were distributed, following a completely randomized design, into groups of three in each pasture. The ruminally cannulated steers were provided the same levels of CDDGS supplementation as in the growing performance study (0%, 0.25%, and 0.50% BW), and they grazed along with the other 40 steers throughout the grazing periods. The dry matter intake, crude protein intake, neutral detergent fiber intake (NDFI), apparent digestibility of dry matter (ADDM), crude protein (ADCP) and neutral detergent fiber (ADNDF) increased with increasing levels of CDDGS supplementation (p<0.05). The ruminal degradation rates of CP (kdCP), NDF (kdNDF) and passage rate (kp) also increased with increasing levels of CDDGS supplementation (p<0.05). Ruminal ammonia nitrogen (NH3-N) and propionate concentrations also increased with increasing levels of CDDGS supplementation (p<0.05). However, acetate concentrations decreased with increasing levels of CDDGS supplementation (p<0.05). Liquid dilution rate increased with increasing levels of CDDGS supplementation but ruminal liquid volume decreased (p<0.05). On the basis of these findings, we can conclude that CDDGS supplementation enhanced the productive performance of cattle grazing native rangeland without negatively affecting forage intake, glucose and urea-nitrogen blood concentrations, ruminal degradation and ruminal fermentation patterns. PMID:26954168

Effect of Supplemental Corn Dried Distillers Grains with Solubles Fed to Beef Steers Grazing Native Rangeland during the Forage Dormant Season.

PubMed

Murillo, M; Herrera, E; Ruiz, O; Reyes, O; Carrete, F O; Gutierrez, H

2016-05-01

Two experiments were conducted to evaluate the effects of the level of corn dry distillers grains with solubles (CDDGS) supplementation on growing performance, blood metabolites, digestion characteristics and ruminal fermentation patterns in steers grazing dormant forage. In Exp. 1, of growth performance, 120 steers (204±5 kg initial body weight [BW]) were distributed randomly into 3 groups (each of 40 steers), which were provided with the following levels of CDDGS supplement: 0%, 0.25%, or 0.50% BW. All groups of steers were grazed for 30 days in each of 3 grazing periods (March, April, and May). Approximately 1,000 ha of the land was divided with electric fencing into 3 equally sized pastures (333 ha in size). Blood samples were collected monthly from 20 steers in each grazing group for analysis of glucose (G), urea-nitrogen (UN) and non-esterified fatty acids. Final BW, average daily gain (ADG) and supplement conversion (CDDGS-C) increased with increasing levels of CDDGS supplementation (p<0.05).The CDDGS supplementation also increased the plasma G and UN concentrations (p<0.05). In Exp. 2, of digestive metabolism, 9 ruminally cannulated steers (BW = 350±3 kg) were distributed, following a completely randomized design, into groups of three in each pasture. The ruminally cannulated steers were provided the same levels of CDDGS supplementation as in the growing performance study (0%, 0.25%, and 0.50% BW), and they grazed along with the other 40 steers throughout the grazing periods. The dry matter intake, crude protein intake, neutral detergent fiber intake (NDFI), apparent digestibility of dry matter (ADDM), crude protein (ADCP) and neutral detergent fiber (ADNDF) increased with increasing levels of CDDGS supplementation (p<0.05). The ruminal degradation rates of CP (kdCP), NDF (kdNDF) and passage rate (kp) also increased with increasing levels of CDDGS supplementation (p<0.05). Ruminal ammonia nitrogen (NH3-N) and propionate concentrations also increased with increasing levels of CDDGS supplementation (p<0.05). However, acetate concentrations decreased with increasing levels of CDDGS supplementation (p<0.05). Liquid dilution rate increased with increasing levels of CDDGS supplementation but ruminal liquid volume decreased (p<0.05). On the basis of these findings, we can conclude that CDDGS supplementation enhanced the productive performance of cattle grazing native rangeland without negatively affecting forage intake, glucose and urea-nitrogen blood concentrations, ruminal degradation and ruminal fermentation patterns.

A Continuation Study Using Sunitinib Malate For Patients Leaving Treatment On A Previous Sunitinib Study.

ClinicalTrials.gov

2015-10-07

Metastatic Breast Cancer [F]; Advanced Breast Cancer; Metastatic Castration Resistant Prostate Cancer; Metastatic Renal Cell Cancer; Non-Small Cell Lung Cancer; Thyroid Cancer; Advanced/Metastatic Non-Small Cell Lung Cancer; Advanced Gastric Cancer; Gastrointestinal Stromal Tumor; Hepatocellular Carcinoma; Pancreatic Islet Cell Carcinoma; Pancreatic Neuroendocrine Tumor

L-malate production by metabolically engineered escherichia coli

DOEpatents

Zhang, Xueli; Wang, Xuan; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

2015-11-17

A process for the production of malic acid in commercially significant quantities from the carbon compounds by genetically modified bacterial strains (GMBS; also referred to as biocatalysts or genetically modified microorganisms) is disclosed. Microorganisms suitable for the production of malic acid can be cultured in one or two-step processes as disclosed herein.

Use of DCPIP in a Colorimetric Method to Investigate Electron Transport in Crude Heart Mitochondrial Extracts.

ERIC Educational Resources Information Center

Myers, Alan

1990-01-01

A method of obtaining a crude mitochondrial extract from sheep and beef heart is described. The respiratory substrates succinate and malate are examined as well as the inhibitory effect of malonate. An interface with a microcomputer for plotting colorimeter results is discussed. (Author/CW)

Carbon Metabolism in Two Species of Pereskia (Cactaceae) 1

PubMed Central

Rayder, Lisa; Ting, Irwin P.

1981-01-01

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor 14CO2 and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO2 uptake pattern indicating C3 carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO2 recycling (“idling”) that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO2 uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested. PMID:16661857

Identification of major allergens in watermelon.

PubMed

Pastor, Carlos; Cuesta-Herranz, Javier; Cases, Barbara; Pérez-Gordo, Marina; Figueredo, Elena; de las Heras, Manuel; Vivanco, Fernando

2009-01-01

Watermelon is a worldwide consumed Cucurbitaceae fruit that can elicit allergic reactions. However, the major allergens of watermelon are not known. The aim of this study is to identify and characterize major allergens in watermelon. Twenty-three patients allergic to watermelon took part in the study. The diagnosis was based on a history of symptoms and positive skin prick-prick tests to watermelon, confirmed by positive open oral challenge testing to watermelon pulp. Allergenic components were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by N-terminal amino acid sequencing and mass spectrometry. Allergens were purified combining several chromatographic steps. Several IgE binding bands (8-120 kDa) were detected in watermelon extract. Three major allergens were identified as malate dehydrogenase (36 kDa), triose phosphate isomerase (28 kDa) and profilin (13 kDa). Purified allergens individually inhibited IgE binding to the whole watermelon extract. All in all these results indicate that malate dehydrogenase, triose phosphate isomerase and profilin are major allergens involved in watermelon allergy. Copyright (C) 2009 S. Karger AG, Basel.

Targeted drug delivery of Sunitinib Malate to tumor blood vessels by cRGD-chiotosan-gold nanoparticles.

PubMed

Saber, Mohaddeseh Mahmoudi; Bahrainian, Sara; Dinarvand, Rassoul; Atyabi, Fatemeh

2017-01-30

The unique characteristics of tumor vasculature represent an attractive strategy for targeted delivery of antitumor and antiangiogenic agents to the tumor. The purpose of this study was to prepare c(RGDfK) labeled chitosan capped gold nanoparticles [cRGD(CS-Au) NPs] as a carrier for selective intracellular delivery of Sunitinib Malate (STB) to the tumor vasculature. cRGD(CS-Au) NPs was formed by electrostatic interaction between cationic CS and anionic AuNPs. cRGD modified CS-Au NPs had a spherical shape with a narrow size distribution. The entrapment efficiency of sunitinib molecule was found to be 45.2%±2.05. Confocal microscopy showed enhanced and selective uptake of cRGD(CS-Au) NPs into MCF-7 and HUVEC cells compared with non-targeted CS-Au NPs. Our results suggest that it may be possible to use cRGD(CS-Au) NPs as a carrier for delivery of anticancer drugs, genes and biomolecules for inhibiting tumor vasculature. Copyright © 2016. Published by Elsevier B.V.

Mitochondrial-morphology-targeted breeding of industrial yeast strains for alcohol fermentation.

PubMed

Kitagaki, Hiroshi

2009-05-29

Since mitochondrial genes are repressed under high glucose and low O2, and these conditions correspond to the conditions in which yeast cells are exposed during alcohol fermentation, the existence and structure of yeast mitochondria during alcohol fermentation have not been elucidated. Yeast mitochondria can be observed throughout brewing of sake (Japanese rice wine) and fragment during brewing. Furthermore, it has been revealed that Fis1 [fission 1 (mitochondrial outer membrane) homologue (Saccharomyces cerevisiae)], which is a transmembrane protein with its C-terminal anchor embedded in the outer membrane of mitochondria, is required for fragmentation of yeast mitochondria during sake brewing. By utilizing this knowledge, a fis1 disruptant of a sake yeast strain has been generated that has a networked mitochondrial structure throughout sake brewing. It transpired that this strain produces a high content of malate, which imparts a crisp acidic taste, during sake brewing. This strategy is a useful and a completely novel strategy towards developing a new yeast strain which produces a high content of malate in sake, and mitochondrial morphology has now emerged as a promising target for the breeding of practical industrial strains.

A time resolved metabolomics study: the influence of different carbon sources during growth and starvation of Bacillus subtilis.

PubMed

Meyer, Hanna; Weidmann, Hendrikje; Mäder, Ulrike; Hecker, Michael; Völker, Uwe; Lalk, Michael

2014-07-01

In its natural environment, the soil, the Gram-positive model bacterium Bacillus subtilis frequently encounters nutrient limitation and other stress factors. Efficient adaptation mechanisms are necessary to cope with this wide range of environmental challenges. The ability to utilize diverse carbon sources represents a key adaptation process that allows B. subtilis to thrive in its natural habitat. To gain a comprehensive insight into the metabolism of B. subtilis, global metabolite analyses were performed during growth with glucose alone or glucose with either malate, fumarate or citrate as carbon/energy sources. Furthermore, to achieve a comprehensive coverage of a wide range of chemically different metabolites, complementary GC-MS, LC-MS and (1)H-NMR analyses were applied. This study reveals that the availability of different carbon sources results in different extracellular metabolite profiles whereas a regulated intracellular metabolite equilibrium was observed. In addition, the typical energy-starvation induced activation of the general stress sigma factor σ(B) was only observed upon entry into the stationary phase with glucose or glucose and malate as carbon sources.

Investigations on the effect of flavonoids from banana, Musa paradisiaca L. on lipid metabolism in rats.

PubMed

Vijayakumar, S; Presannakumar, G; Vijayalakshmi, N R

2009-01-01

Oral administration of flavonoids extracted from unripe fruits of Musa paradisiaca showed significant hypolipidemic activities in male rats (Sprague Dawley strain) at a dose of 1 mg/100 g body weight (BW)/day. Concentrations of cholesterol, phospholipids, free fatty acids, and triglycerides showed significant decrease in the serum, liver, kidney, and brain of experimental animals. HMG CoA reductase activity was found to be enhanced, while activities of glucose-6-phosphate dehydrogenase and malate dehydrogenase were significantly reduced. Activities of lipoprotein lipase and plasma LCAT showed significant enhancement. A significant increase in the concentrations of hepatic and fecal bile acids and fecal neutral sterols was also observed indicating a higher rate of degradation of cholesterol. The present study indicates that although there is an increase in the rate of synthesis of cholesterol in the liver, the process of degradation exceeds the rate of synthesis.

[Levels of unified metabolites and thyroid hormones in blood and oral fluid of children with minimal brain dysfunction].

PubMed

Gil'miiarova, F N; Pervova, Iu V; Radomskaia, V M; Gergel', N I; Tarasova, S V

2004-01-01

Minimal brain dysfunctions in children with various perinatal complications are accompanied by metabolic imbalance manifested by decreased total protein content, the tendency to reduced triglycerides, increased cholesterol concentrations in the oral fluid, the trend to hypoproteinaemia, hypoglycaemia, hypotriglyceridaemia. The most significant changes in the redox systems alpha-ketoglutarate-glutamate, oxaloacetate-malate, pyruvate-lactate, dioxyacetone phosphate-alpha-glycerophosphate in biological fluids were revealed in cases of antenatal alcoholisation. A certain correlation was found between anemia in pregnant women and hypothyroidal background in children. In addition, a high level of free and total thyroxine, that of total triiodthyronine were found in the oral fluid. Hypophysis--thyroid dysregulation in children with minimal brain dysfunction associated with gestosis in their mothers during pregnancy, was manifested by decreased content of total and free T4 and T3 in blood serum and increased level of the thyroid-stimulating hormone.

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts.

PubMed

Crawford, N A; Droux, M; Kosower, N S; Buchanan, B B

1989-05-15

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.

Molecular and physiological strategies to increase aluminum resistance in plants.

PubMed

Inostroza-Blancheteau, Claudio; Rengel, Zed; Alberdi, Miren; de la Luz Mora, María; Aquea, Felipe; Arce-Johnson, Patricio; Reyes-Díaz, Marjorie

2012-03-01

Aluminum (Al) toxicity is a primary limitation to plant growth on acid soils. Root meristems are the first site for toxic Al accumulation, and therefore inhibition of root elongation is the most evident physiological manifestation of Al toxicity. Plants may resist Al toxicity by avoidance (Al exclusion) and/or tolerance mechanisms (detoxification of Al inside the cells). The Al exclusion involves the exudation of organic acid anions from the root apices, whereas tolerance mechanisms comprise internal Al detoxification by organic acid anions and enhanced scavenging of free oxygen radicals. One of the most important advances in understanding the molecular events associated with the Al exclusion mechanism was the identification of the ALMT1 gene (Al-activated malate transporter) in Triticum aestivum root cells, which codes for a plasma membrane anion channel that allows efflux of organic acid anions, such as malate, citrate or oxalate. On the other hand, the scavenging of free radicals is dependent on the expression of genes involved in antioxidant defenses, such as peroxidases (e.g. in Arabidopsis thaliana and Nicotiana tabacum), catalases (e.g. in Capsicum annuum), and the gene WMnSOD1 from T. aestivum. However, other recent findings show that reactive oxygen species (ROS) induced stress may be due to acidic (low pH) conditions rather than to Al stress. In this review, we summarize recent findings regarding molecular and physiological mechanisms of Al toxicity and resistance in higher plants. Advances have been made in understanding some of the underlying strategies that plants use to cope with Al toxicity. Furthermore, we discuss the physiological and molecular responses to Al toxicity, including genes involved in Al resistance that have been identified and characterized in several plant species. The better understanding of these strategies and mechanisms is essential for improving plant performance in acidic, Al-toxic soils.

Oxaloacetate-to-malate conversion by mineral photoelectrochemistry: implications for the viability of the reductive tricarboxylic acid cycle in prebiotic chemistry

NASA Astrophysics Data System (ADS)

Guzman, Marcelo I.; Martin, Scot T.

2008-10-01

The carboxylic acids produced by the reductive tricarboxylic acid (rTCA) cycle are possibly a biosynthetic core of initial life, although several steps such as the reductive kinetics of oxaloacetate (OAA) to malate (MA) are problematic by conventional chemical routes. In this context, we studied the kinetics of this reaction as promoted by ZnS mineral photoelectrochemistry. The quantum efficiency φMA of MA production from the photoelectrochemical reduction of OAA followed φMA=0.13 [OAA] (2.1×10-3+[OAA])-1 and was independent of temperature (5 to 50°C). To evaluate the importance of this forward rate under a prebiotic scenario, we also studied the temperature-dependent rate of the backward thermal decarboxylation of OAA to pyruvate (PA), which followed an Arrhenius behavior as log (k-2)=11.74 4956/T, where k-2 is in units of s-1. These measured rates were employed in conjunction with the indirectly estimated carboxylation rate of PA to OAA to assess the possible importance of mineral photoelectrochemistry in the conversion of OAA to MA under several scenarios of prebiotic conditions on early Earth. As an example, our analysis shows that there is 90% efficiency with a forward velocity of 3 yr/cycle for the OAA→MA step of the rTCA cycle at 280 K. Efficiency and velocity both decrease for increasing temperature. These results suggest high viability for mineral photoelectrochemistry as an enzyme-free engine to drive the rTCA cycle through the early aeons of early Earth, at least for the investigated OAA→MA step.

Mitochondrial phenotypic flexibility enhances energy savings during winter fast in king penguin chicks.

PubMed

Monternier, Pierre-Axel; Marmillot, Vincent; Rouanet, Jean-Louis; Roussel, Damien

2014-08-01

Energy conservation is a key priority for organisms that live in environments with seasonal shortages in resource supplies or that spontaneously fast during their annual cycle. The aim of this study was to determine whether the high fasting endurance of winter-acclimatized king penguin chicks (Aptenodytes patagonicus) is associated with an adjustment of mitochondrial bioenergetics in pectoralis muscle, the largest skeletal muscle in penguins. The rates of mitochondrial oxygen consumption, and ATP synthesis and mitochondrial efficiency (ATP/O ratio) were measured in winter-acclimatized chicks. We used pyruvate/malate and palmitoyl-l-carnitine/malate as respiratory substrates and results from naturally fasted chicks were compared to experimentally re-fed chicks. Bioenergetics analysis of pectoralis muscle revealed that mitochondria are on average 15% more energy efficient in naturally fasted than in experimentally fed chicks, indicating that fasted birds consume less nutrients to sustain their energy-demanding processes. We also found that moderate reductions in temperature from 38°C to 30°C further increase by 23% the energy coupling efficiency at the level of mitochondria, suggesting that king penguin chicks realize additional energy savings while becoming hypothermic during winter. It has been calculated that this adjustment of mitochondrial efficiency in skeletal muscle may contribute to nearly 25% of fasting-induced reduction in mass-specific metabolic rate measured in vivo. The present study shows that the regulation of mitochondrial efficiency triggers the development of an economical management of resources, which would maximize the conservation of endogenous fuel stores by decreasing the cost of living in fasted winter-acclimatized king penguin chicks. © 2014. Published by The Company of Biologists Ltd.

Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

PubMed

Yoshida, Keisuke; Hisabori, Toru

2016-06-01

Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance. Copyright © 2016 Elsevier B.V. All rights reserved.

Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered Patterns of Aluminum Accumulation and Organic Acid Release from Roots1

PubMed Central

Larsen, Paul B.; Degenhardt, Jörg; Tai, Chin-Yin; Stenzler, Laura M.; Howell, Stephen H.; Kochian, Leon V.

1998-01-01

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27). PMID:9576769

Supplemental vitamin D increases serum cytokines in those with initially low 25-hydroxyvitamin D: a randomized, double blind, placebo-controlled study.

PubMed

Barker, Tyler; Rogers, Victoria E; Levy, Mark; Templeton, Jenna; Goldfine, Howard; Schneider, Erik D; Dixon, Brian M; Henriksen, Vanessa T; Weaver, Lindell K

2015-02-01

The purpose of this study was to determine if vitamin D status before supplementation influences the cytokine response after supplemental vitamin D. Forty-six reportedly healthy adults (mean(SD); age, 32(7) y; body mass index (BMI), 25.3(4.5) kg/m(2); serum 25-hydroxyvitamin D (25(OH)D), 34.8(12.2) ng/mL) were randomly assigned (double blind) to one of three groups: (1) placebo (n=15), or supplemental vitamin D (cholecalciferol) at (2) 4000 (n=14) or (3) 8000IU (n=17). Supplements were taken daily for 35days. Fasting blood samples were obtained before (Baseline, Bsl) and 35-days after (35-d) supplementation. Serum 25(OH)D, 1,25-dihydroxyvitamin D (1,25(OH)D), cytokines, and intact parathyroid hormone with calcium were measured in each blood sample. Supplemental vitamin D increased serum 25(OH)D (4000IU, ≈29%; 8000IU, ≈57%) and 1,25(OH)D (4000IU, ≈12%; 8000IU, ≈38%) without altering intact parathyroid hormone or calcium. The vitamin D metabolite increases in the supplemental vitamin D groups (n=31) were dependent on initial levels as serum 25(OH)D (r=-0.63, p<0.05) and 1,25(OH)D (r=-0.45, p<0.05) at Bsl correlated with their increases after supplementation. Supplemental vitamin D increased interferon (IFN)-γ and interleukin (IL)-10 in subjects that were vitamin D insufficient (serum 25(OH)D<29ng/mL) compared to sufficient (serum 25(OH)D⩾30ng/mL) at Bsl. We conclude that supplemental vitamin D increase a pro- and anti-inflammatory cytokine in those with initially low serum 25(OH)D. Copyright © 2014 Elsevier Ltd. All rights reserved.

Crassulacean acid metabolism-cycling in Euphorbia milii.

PubMed

Herrera, Ana

2013-01-01

Crassulacean acid metabolism (CAM) occurs in many Euphorbiaceae, particularly Euphorbia, a genus with C3 and C4 species as well. With the aim of contributing to our knowledge of the evolution of CAM in this genus, this study examined the possible occurrence of CAM in Euphorbia milii, a species with leaf succulence and drought tolerance suggestive of this carbon fixation pathway. Leaf anatomy consisted of a palisade parenchyma, a spongy parenchyma and a bundle sheath with chloroplasts, which indicates the possible functioning of C2 photosynthesis. No evidence of nocturnal CO2 fixation was found in plants of E. milii either watered or under drought; watered plants had a low nocturnal respiration rate (R). After 12 days without watering, the photosynthetic rate (P N) decreased 85 % and nocturnal R was nearly zero. Nocturnal H(+) accumulation (ΔH(+)) in watered plants was 18 ± 2 (corresponding to malate) and 18 ± 4 (citrate) μmol H(+) (g fresh mass)(-1). Respiratory CO2 recycling through acid synthesis contributed to a night-time water saving of 2 and 86 % in watered plants and plants under drought, respectively. Carbon isotopic composition (δ(13)C) was -25.2 ± 0.7 ‰ in leaves and -24.7 ± 0.1 ‰ in stems. Evidence was found for the operation of weak CAM in E. milii, with statistically significant ΔH(+), no nocturnal CO2 uptake and values of δ(13)C intermediate between C3 and constitutive CAM plants; ΔH(+) was apparently attributable to both malate and citrate. The results suggest that daily malate accumulation results from recycling of part of the nocturnal respiratory CO2, which helps explain the occurrence of an intermediate value of leaf δ(13)C. Euphorbia milii can be considered as a CAM-cycling species. The significance of the operation of CAM-cycling in E. milii lies in water conservation, rather than carbon acquisition. The possible occurrence of C2 photosynthesis merits research.

To supplement or not to supplement: a metabolic network framework for human nutritional supplements.

PubMed

Nogiec, Christopher D; Kasif, Simon

2013-01-01

Flux balance analysis and constraint based modeling have been successfully used in the past to elucidate the metabolism of single cellular organisms. However, limited work has been done with multicellular organisms and even less with humans. The focus of this paper is to present a novel use of this technique by investigating human nutrition, a challenging field of study. Specifically, we present a steady state constraint based model of skeletal muscle tissue to investigate amino acid supplementation's effect on protein synthesis. We implement several in silico supplementation strategies to study whether amino acid supplementation might be beneficial for increasing muscle contractile protein synthesis. Concurrent with published data on amino acid supplementation's effect on protein synthesis in a post resistance exercise state, our results suggest that increasing bioavailability of methionine, arginine, and the branched-chain amino acids can increase the flux of contractile protein synthesis. The study also suggests that a common commercial supplement, glutamine, is not an effective supplement in the context of increasing protein synthesis and thus, muscle mass. Similar to any study in a model organism, the computational modeling of this research has some limitations. Thus, this paper introduces the prospect of using systems biology as a framework to formally investigate how supplementation and nutrition can affect human metabolism and physiology.

Effect of hormonal synchronisation and/or short-term supplementation with maize on follicular dynamics and hormone profiles in goats during the non-breeding season.

PubMed

Nogueira, D M; Cavalieri, J; Fitzpatrick, L A; Gummow, B; Blache, D; Parker, A J

2016-08-01

This study aimed to evaluate the reproductive response of anoestrous goats that were either hormonally treated and/or supplemented with maize for 9days to determine which treatment combination was the most effective in enhancing follicular development and ovulation rate, and whether these responses were associated with increases in metabolic hormones. The experiment was carried out using 28 does, using a 2×2 factorial design with seven does in each group to test the effect of synchronisation of oestrus, supplementation with maize and their interactions. Synchronisation of oestrous cycles (P<0.001) but not supplementation with maize or the interaction between the two (P>0.05) increased the number of codominant follicles, the diameter of the largest follicle on Day 9 and growth rate of follicles during the period of supplementation. Compared with non-supplemented animals, supplementation with maize increased the total number of follicles observed between Days 7 and 9 (P=0.039). In addition, nutritional supplementation with maize in combination with synchronisation of oestrus increased the ovulation rate by 43% (P=0.074). Interactions between time and supplementation with maize showed that plasma concentrations of insulin, leptin and IGF-1 were greater in does supplemented with maize compared with non-supplemented does (P<0.001). The findings show that hormonal synchronisation had the most influence on modifying follicular development and ovulation in anoestrous goats. Supplementation with maize increased the concentrations of insulin, leptin and IGF-1, which could potentially modify the sensitivity of follicles to gonadotrophins and reduce rates of atresia. Copyright © 2016 Elsevier B.V. All rights reserved.

Milk production and nutrient digestibility responses to increasing levels of stearic acid supplementation of dairy cows.

PubMed

Boerman, J P; de Souza, J; Lock, A L

2017-04-01

The objective of our study was to evaluate the dose-response effects of a stearic acid (C18:0)-enriched supplement on nutrient digestibility, production responses, and the maximum amount of C18:0 that can be incorporated into the milk fat of dairy cows. Multiparous Holstein cows (n = 32; 145 ± 66 d in milk) with a wide range in milk yield (30 to 70 kg/d) were blocked by milk yield and assigned to replicated 4 × 4 Latin squares. Treatments were diets supplemented with a C18:0-enriched supplement (SA; 93% C18:0) at 0, 0.80, 1.50, or 2.30% of diet dry matter (DM). Periods were 21 d with the final 5 d used for data and sample collection. Dry matter intake increased linearly as SA supplementation increased. Supplementation of SA had no effect on the yield of milk or milk components. Due to the increase in DM intake, SA linearly reduced the ratio of energy-corrected milk to DM intake. Supplementation of SA did not affect body weight. Increasing SA reduced digestibility of 16-carbon, 18-carbon, and total fatty acids (FA), with the reduction in digestibility of 18-carbon FA being approximately 30 percentage units from the 0.0 to 2.30% SA supplemented diets. Supplementation of SA linearly increased concentrations of preformed milk fatty acids (FA) but did not affect the yield of preformed milk FA. Yields of C18:0 plus cis-9 C18:1 were increased by SA supplementation; however, the increase from 0 to 2.3% SA was only 16 g/d. The concentration and yield of de novo and 16-carbon milk FA were unaffected by SA supplementation. In conclusion, increasing doses of SA decreased FA digestibility and had little effect on production parameters. Although SA increased the yield of C18:0 and cis-9 C18:1 in milk fat, it had no overall effect on milk fat yield. The lack of production responses to a C18:0-enriched fat supplement was most likely associated with the marked decrease in FA digestibility. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Fermentation performance of lager yeast in high gravity beer fermentations with different sugar supplementations.

PubMed

Lei, Hongjie; Xu, Huaide; Feng, Li; Yu, Zhimin; Zhao, Haifeng; Zhao, Mouming

2016-11-01

The effects of glucose, sucrose and maltose supplementations on the fermentation performance and stress tolerance of lager yeast (Saccharomyces pastorianus) during high gravity (18°P) and very high gravity (24°P) fermentations were studied. Results showed that throughout 18°P wort fermentation, fermentation performance of lager yeast was significantly improved by glucose or sucrose supplementation, compared with maltose supplementation, especially for sucrose supplementation increasing wort fermentability and ethanol production by 6% and 8%, respectively. However, in the later stage of 24°P wort fermentation, fermentation performance of lager yeast was dramatically improved by maltose supplementation, which increased wort fermentability and ethanol production by 14% and 10%, respectively, compared with sucrose supplementation. Furthermore, higher HSP12 expression level and more intracellular trehalose accumulation in yeast cells were observed by maltose supplementation with increase of the wort gravity from 18°P to 24°P, indicating higher stress response of yeast cells. The excretion of Gly and Ala, and the absorption of Pro in the later stage of fermentation were promoted by maltose supplementation. In addition, with increase of the wort gravity from 18°P to 24°P, higher alcohols level was decreased with maltose supplementation, while esters formation was increased significantly with glucose supplementation. This study suggested that the choice of optimal fermentable sugars maintaining better fermentation performance of lager yeast should be based on not only strain specificity, but also wort gravity. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Omega-3 supplementation improves cognition and modifies brain activation in young adults.

PubMed

Bauer, Isabelle; Hughes, Matthew; Rowsell, Renee; Cockerell, Robyn; Pipingas, Andrew; Crewther, Sheila; Crewther, David

2014-03-01

The current study aimed to investigate the effects of eicosapentaenoic acid (EPA)-rich and docosahexaenoic acid (DHA)-rich supplementations on cognitive performance and functional brain activation. A double-blind, counterbalanced, crossover design, with a 30-day washout period between two supplementation periods (EPA-rich and DHA-rich) was employed. Functional magnetic resonance imaging scans were obtained during performance of Stroop and Spatial Working Memory tasks prior to supplementation and after each 30-day supplementation period. Both supplementations resulted in reduced ratio of arachidonic acid to EPA levels. Following the EPA-rich supplementation, there was a reduction in functional activation in the left anterior cingulate cortex and an increase in activation in the right precentral gyrus coupled with a reduction in reaction times on the colour-word Stroop task. By contrast, the DHA-rich supplementation led to a significant increase in functional activation in the right precentral gyrus during the Stroop and Spatial Working Memory tasks, but there was no change in behavioural performance. By extending the theory of neural efficiency to the within-subject neurocognitive effects of supplementation, we concluded that following the EPA-rich supplementation, participants' brains worked 'less hard' and achieved a better cognitive performance than prior to supplementation. Conversely, the increase in functional activation and lack of improvement in time or accuracy of cognitive performance following DHA-rich supplementation may indicate that DHA-rich supplementation is less effective than EPA-rich supplementation in enhancing neurocognitive functioning after a 30-day supplementation period in the same group of individuals.

Meta-analysis of the influence of Saccharomyces cerevisiae supplementation on ruminal parameters and milk production of ruminants.

PubMed

Desnoyers, M; Giger-Reverdin, S; Bertin, G; Duvaux-Ponter, C; Sauvant, D

2009-04-01

The effects of yeast supplementation on intake, production, and rumen fermentation characteristics have been widely studied, but results are inconsistent between different studies. A quantitative meta-analysis was applied to 110 papers, 157 experiments, and 376 treatments dealing with yeast supplementation in ruminants. The objective was first to highlight the major quantitative effects of live yeast supplementation on intake, rumen fermentation, and milk production, and second, to identify major differences in experimental conditions between studies that can affect the response to treatment. Some of these experimental conditions are referred to as interfering factors. Yeast supplementation increased rumen pH (+0.03 on average) and rumen volatile fatty acid concentration (+2.17 mM on average), tended to decrease rumen lactic acid concentration (-0.9 mM on average), and had no influence on acetate-to-propionate ratio. Total-tract organic matter digestibility was also increased by yeast supplementation (+0.8% on average). Yeast supplementation increased dry matter intake (DMI; +0.44 g/kg of body weight; BW), milk yield (+1.2 g/kg of BW), and tended to increase milk fat content (+0.05%), but had no influence on milk protein content. Dose effects of yeast supplementation, expressed as log(10) [1+(cfu per 100 kg of BW)], globally confirmed the qualitative effects observed in the first analysis. The positive effect of yeast supplementation on rumen pH increased with the percentage of concentrate in the diet and with the DMI level. It was negatively correlated with the level of dietary neutral detergent fiber (NDF). The positive effect of yeast supplementation on rumen volatile fatty acid concentration increased with DMI and crude protein levels. The positive effect of yeast supplementation on organic matter digestibility increased with the percentage of concentrate and NDF in the diet. The negative effect of yeast supplementation on lactic acid concentration tended to decrease when the DMI level and the percentage of concentrate in the diet increased. The effects of interfering factors were globally similar when either dose effect or qualitative effect of yeast was taken into account. Although rumen fermentation efficiency per se was not measured, these results suggest an improvement in rumen fermentation by yeast supplementation. This effect could, however, be modulated by several different factors such as DMI, percentage of concentrate or NDF in the diet, or species.

Integration between Glycolysis and Glutamate-Glutamine Cycle Flux May Explain Preferential Glycolytic Increase during Brain Activation, Requiring Glutamate

PubMed Central

Hertz, Leif; Chen, Ye

2017-01-01

The 1988 observation by Fox et al. (1988) that brief intense brain activation increases glycolysis (pyruvate formation from glucose) much more than oxidative metabolism has been abundantly confirmed. Specifically glycolytic increase was unexpected because the amount of ATP it generates is much smaller than that formed by subsequent oxidative metabolism of pyruvate. The present article shows that preferential glycolysis can be explained by metabolic processes associated with activation of the glutamate-glutamine cycle. The flux in this cycle, which is essential for production of transmitter glutamate and GABA, equals 75% of brain glucose utilization and each turn is associated with utilization of ~1 glucose molecule. About one half of the association between cycle flux and glucose metabolism occurs during neuronal conversion of glutamine to glutamate in a process similar to the malate-aspartate shuttle (MAS) except that glutamate is supplied from glutamine, not formed from α-ketoglutarate (αKG) as during operation of conventional MAS. Regular MAS function is triggered by one oxidative process in the cytosol during glycolysis causing NAD+ reduction to NADH. Since NADH cannot cross the mitochondrial membrane (MEM) for oxidation NAD+ is re-generated by conversion of cytosolic oxaloacetate (OAA) to malate, which enters the mitochondria for oxidation and in a cyclic process regenerates cytosolic OAA. Therefore MAS as well as the “pseudo-MAS” necessary for neuronal glutamate formation can only operate together with cytosolic reduction of NAD+ to NADH. The major process causing NAD+ reduction is glycolysis which therefore also must occur during neuronal conversion of glutamine to glutamate and may energize vesicular glutamate uptake which preferentially uses glycolytically derived energy. Another major contributor to the association between glutamate-glutamine cycle and glucose utilization is the need for astrocytic pyruvate to generate glutamate. Although some oxidative metabolism occurs during glutamate formation it is only one half of that during normal tricarboxylic acid (TCA) cycle function. Glutamate’s receptor stimulation leads to potassium ion (K+) release and astrocytic uptake, preferentially fueled by glycolysis and followed by release and neuronal re-accumulation. The activation-induced preferential glycolysis diminishes with continued activation and is followed by an increased ratio between oxidative metabolism and glycolysis, reflecting oxidation of generated glutamate and accumulated lactate. PMID:28890689

[CAM in Tillandsia usneoides: Studies on the pathway of carbon and the dependency of CO2-exchange on light intensity, temperature and water content of the plant].

PubMed

Kluge, M; Lange, O L; Eichmann, M V; Schmid, R

1973-12-01

Tillandsia usneoides, in the common sense a non-succulent plant, exhibits CO2 exchange characterized by net CO2 dark fixation during the night and depression of CO2 exchange during the day. Malate has been demonstrated to accumulate during CO2 dark fixation and to be converted to carbohydrates in light. Thus, T. usneoides exhibits CAM like typical succulents.Net CO2 uptake during the day is increased with net CO2 output being suppressed in duration of time and extent when light intensity increases. Furthermore, a slight increase in CO2 fixation during the following night can be observed if the plants were treated with high light intensity during the previous day.Curves of CO2 exchange typical for CAM are obtained if T. usneoides is kept at 15°C and 20°C. Lower temperature tend to increase CO2 uptake during the day and to inhibit CO2 dark fixation. Temperatures higher than 20°C favour loss of CO2 by respiration, which becomes apparent during the whole day and night at 30°C and higher temperatures. Thus, T. usneoides gains carbon only at temperatures well below 25°C.Net CO2 uptake during the day occurs only in moist plant material and is inhibited in plants cept under water stress conditions. However, CO2 uptake during the night is clearly favoured if the plants dry out. Therefore dry plants gain more carbon than moist ones.Curves of CO2 exchange typical for CAM were also obtained with 13 other species of the genus Tillandsia.The exhibition of CAM by the non-succulent T. usneoides calls for a new definition of the term "succulence" if it is to remain useful in characterizing this metabolic pathway. Because CO2-fixing cells of T. usneoides possess relatively large vacuoles and are relatively poor in chloroplasts, they resembles the assimilatory cells of typical CAM-exhibiting succulents. Therefore, if "succulence" only means the capacity of big vacuoles to store malate, the assimilatory cells in T. usneoides are succulent. It seems to be useful to investigate parameters which would allow a definition of the term "succulence" on the level of the cell rather than on the level of the whole plant or plant organs.

Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux

USDA-ARS?s Scientific Manuscript database

Aluminum (Al) toxicity is a primary limitation to crop productivity on acid soils throughout the plant. Root efflux of organic acid anions constitutes a mechanism by which plants cope with toxic aluminum (Al) ions on acid soils. In this study, we have characterized ZmALMT2 (a member of aluminum-acti...

Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate.

PubMed Central

Cevallos, M A; Encarnación, S; Leija, A; Mora, Y; Mora, J

1996-01-01

Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen. PMID:8626293

Mechanisms for hydrogen production by different bacteria during mixed-acid and photo-fermentation and perspectives of hydrogen production biotechnology.

PubMed

Trchounian, Armen

2015-03-01

H2 has a great potential as an ecologically-clean, renewable and capable fuel. It can be mainly produced via hydrogenases (Hyd) by different bacteria, especially Escherichia coli and Rhodobacter sphaeroides. The operation direction and activity of multiple Hyd enzymes in E. coli during mixed-acid fermentation might determine H2 production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating the activity of different Hyd enzymes is an effective way to enhance H2 production by E. coli in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H2 production. Mixed carbon (sugar and glycerol) utilization studies enlarge the kind of organic wastes used in biotechnology. During photo-fermentation under limited nitrogen conditions, H2 production by Rh. sphaeroides is observed when carbon and nitrogen sources are supplemented. The relationship of H2 production with H(+) transport across the membrane and membrane-associated ATPase activity is shown. On the other hand, combination of carbon sources (succinate, malate) with different nitrogen sources (yeast extract, glutamate, glycine) as well as different metal (Fe, Ni, Mg) ions might regulate H2 production. All these can enhance H2 production yield by Rh. sphaeroides in biotechnology Finally, two of these bacteria might be combined to develop and consequently to optimize two stages of H2 production biotechnology with high efficiency transformation of different organic sources.

Time-divided ingestion pattern of casein-based protein supplement stimulates an increase in fat-free body mass during resistance training in young untrained men.

PubMed

Burk, Andres; Timpmann, Saima; Medijainen, Luule; Vähi, Mare; Oöpik, Vahur

2009-06-01

We hypothesized that during prolonged resistance training, time-divided ingestion pattern of casein-based protein supplement is of superior efficiency in comparison with the ingestion of the same supplement immediately before each training session. In a crossover study, 13 men aged 18 to 19 years were evaluated during 2 well-controlled, 8-week training and supplementation periods. In the time-focused supplementation regimen (TFR), the subjects consumed the supplement in the morning and in the afternoon, immediately before the training session. Time-divided supplementation regimen (TDR) included 1 morning dose, whereas the second dose was ingested in the evening, 5 hours after training. The daily dose of the supplement contained approximately 70 g of protein (82% casein) and less than 1 g of carbohydrate and fat. Body mass, body composition (dual-energy x-ray absorptiometry scanned), and one-repetition maximum (1RM) for bench press and squat were determined at the beginning and at the end of both 8-week training and supplementation periods. Training produced a significant increase in 1RM strength both in the bench press (9.4% and 7.2%) and the squat exercise (10.7% and 17.8%) in the TFR and TDR, respectively, with no differences between the supplementation regimens. Fat-free mass increased from 62.4 +/- 1.2 to 63.5 +/- 1.3 kg (P = .046) with TDR, whereas no change was evident with TFR. The increase in 1RM strength in the squat exercise was related to the increase in fat-free mass in TDR (r = 0.569; P = .041). These findings may have practical implications for the timing of ingestion of protein supplements to enhance the efficacy of resistance training.

Increased natural reproduction and genetic diversity one generation after cessation of a steelhead trout (Oncorhynchus mykiss) conservation hatchery program.

PubMed

Berejikian, Barry A; Van Doornik, Donald M

2018-01-01

Spatial and temporal fluctuations in productivity and abundance confound assessments of captive propagation programs aimed at recovery of Threatened and Endangered populations. We conducted a 17 year before-after-control-impact experiment to determine the effects of a captive rearing program for anadromous steelhead trout (Oncorhynchus mykiss) on a key indicator of natural spawner abundance (naturally produced nests or 'redds'). The supplemented population exhibited a significant (2.6-fold) increase in redd abundance in the generation following supplementation. Four non-supplemented (control) populations monitored over the same 17 year period exhibited stable or decreasing trends in redd abundance. Expected heterozygosity in the supplemented population increased significantly. Allelic richness increased, but to a lesser (non-significant) degree. Estimates of the effective number of breeders increased from a harmonic mean of 24.4 in the generation before supplementation to 38.9 after supplementation. Several non-conventional aspects of the captive rearing program may have contributed to the positive response in the natural population.

Bodybuilding supplementation and tooth decay.

PubMed

Ali, M S; Batley, H; Ahmed, F

2015-07-10

Supplementation is a key component in bodybuilding and is increasingly being used by amateur weight lifters and enthusiasts to build their ideal bodies. Bodybuilding supplements are advertised to provide nutrients needed to help optimise muscle building but they can contain high amounts of sugar. Supplement users are consuming these products, while not being aware of their high sugar content, putting them at a higher risk of developing dental caries. It is important for dental professionals to recognise the increased risk for supplement users and to raise awareness, provide appropriate preventative advice and be knowledgeable of alternative products to help bodybuilders reach their goals, without increasing the risk of dental caries.

Supplementation of Flemingia macrophylla and cassava foliage as a rumen enhancer on fermentation efficiency and estimated methane production in dairy steers.

PubMed

Phesatcha, Burarat; Wanapat, Metha; Phesatcha, Kampanat; Ampapon, Thiwakorn; Kang, Sungchhang

2016-10-01

Four rumen-fistulated dairy steers, 3 years old with 180 ± 15 kg body weight (BW), were randomly assigned according to a 4 × 4 Latin square design to investigate on the effect of Flemingia macrophylla hay meal (FMH) and cassava hay meal (CH) supplementation on rumen fermentation efficiency and estimated methane production. The treatments were as follows: T1 = non-supplement, T2 = CH supplementation at 150 g/head/day, T3 = FMH supplementation at 150 g/head/day, and T4 = CH + FMH supplementation at 75 and 75 g/head/day. All steers were fed rice straw ad libitum and concentrate was offered at 0.5 % of BW. Results revealed that supplementation of CH and/or FMH did not affect on feed intake (P > 0.05) while digestibility of crude protein and neutral detergent fiber were increased especially in steers receiving FMH and CH+FMH (P < 0.05). Ruminal pH, temperature, and blood urea nitrogen were similar among treatments while ammonia nitrogen was increased in all supplemented groups (P < 0.05). Furthermore, propionic acid (C3) was increased while acetic acid (C2), C2:C3 ratio, and estimated methane production were decreased by dietary treatments. Protozoa and fungi population were not affected by dietary supplement while viable bacteria count increased in steers receiving FMH. Supplementation of FMH and/or FMH+CH increased microbial crude protein and efficiency of microbial nitrogen supply. This study concluded FMH (150 g/head/day) and/or CH+FMH (75 and 75 g/head/day) supplementation could be used as a rumen enhancer for increasing nutrient digestibility, rumen fermentation efficiency, and microbial protein synthesis while decreasing estimated methane production without adverse effect on voluntary feed intake of dairy steers fed rice straw.

[Effect of L-arginine and the nitric oxide synthase blocker L-NNA on calcium capacity in rat liver mitochondria with differing resistance to hypoxia].

PubMed

Kurhaliuk, N M; Ikkert, O V; Vovkanych, L S; Horyn', O V; Hal'kiv, M O; Hordiĭ, S K

2001-01-01

The effect of L-arginine and blockator of nitric oxide synthase L-NNA on processes of calcium mitochondrial capacity in liver with different resistance to hypoxia in the experiments with Wistar rats has been studied using the followrng substrates of energy support: succinic, alpha-ketoglutaric acids, alpha-ketolutarate and inhibitor succinatedehydrogenase malonate. As well we used substrates mixtures combination providing for activation of aminotransferase mechanism: glutamate and piruvate, glutamate and malate. It has been shown that L-arginine injection increases calcium mitochondrial capacity of low resistant rats using as substrates the succinate and alpha-ketoglutarate to control meanings of high resistance rats. Effects of donors nitric oxide on this processes limit NO-synthase inhibitor L-NNA.

Proteomic Analysis of Responsive Proteins Induced in Japanese Birch Plantlet Treated with Salicylic Acid

PubMed Central

Suzuki, Hiromu; Takashima, Yuya; Ishiguri, Futoshi; Yoshizawa, Nobuo; Yokota, Shinso

2014-01-01

The present study was performed to unravel the mechanisms of systemic acquired resistance (SAR) establishment and resistance signaling pathways against the canker-rot fungus (Inonotus obliquus strain IO-U1) infection in Japanese birch plantlet No.8. Modulation of protein-profile induced by salicylic acid (SA)-administration was analyzed, and SA-responsive proteins were identified. In total, 5 specifically expressed, 3 significantly increased, and 3 significantly decreased protein spots were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the sequence tag method. These proteins were malate dehydrogenase, succinate dehydrogenase, phosphoglycerate kinase, diaminopimalate decarboxylase, arginase, chorismate mutase, cyclophilin, aminopeptidase, and unknown function proteins. These proteins are considered to be involved in SAR-establishment mechanisms in the Japanese birch plantlet No 8. PMID:28250384

Effects of dietary protein levels and 2-methylbutyrate on ruminal fermentation, nutrient degradability, bacterial populations and urinary purine derivatives in Simmental steers.

PubMed

Wang, C; Liu, Q; Guo, G; Huo, W J; Pei, C X; Zhang, S L; Yang, W Z

2018-06-01

The objective of this study was to evaluate the effects of dietary crude protein (CP) levels and 2-methylbutyrate (MB) supplementation on ruminal fermentation, bacterial populations, microbial enzyme activity and urinary excretion of purine derivatives (PD) in Simmental steers. Eight ruminally cannulated Simmental steers, averaging 18 months of age and 465 ± 8.6 kg of body weight (BW), were used in a replicated 4 × 4 Latin square design by a 2 × 2 factorial arrangement. Low protein (98.5 g CP/kg dry matter [LP] or high protein (128.7 g CP/kg dry matter [HP]) diets were fed with MB supplementation (0 g [MB-] or 16.8 g steer -1  day -1 [MB+]). Steers were fed a total mixed ration with dietary corn straw to concentrate ratio of 50:50 (dry matter [DM] basis). The CP × MB interaction was observed for ruminal total VFA, molar proportions of acetate and propionate, acetate to propionate ratio, ammonia-N, effective degradability of neutral detergent fibre (NDF) and CP, microbial enzyme activity, bacterial populations and total PD excretion (p < .05). Ruminal pH decreased (p < .05), but ruminal total VFA concentration increased (p < .05) with increasing dietary CP level or MB supplementation. Acetate molar proportion increased (p = .043) with MB supplementation, but was not affected by dietary CP level. Propionate molar proportion decreased (p < .05) with increasing dietary CP level or MB supplementation. Consequently, acetate-to-propionate ratio increased (p = .001) with MB supplementation, but was not affected by dietary CP level. Ruminal ammonia-N content increased (p = .034) with increasing dietary CP level, but decreased (p = .012) with MB supplementation. The effective degradability of NDF and CP increased (p < .05) with increasing dietary CP level or MB supplementation. Microbial enzyme activity, bacterial populations and total PD excretion also increased (p < .05) with increasing dietary CP level or MB supplementation. The results indicated that ruminal fermentation, nutrient degradability, microbial enzyme activity, ruminal bacterial populations and microbial protein synthesis improved with increasing dietary CP level or MB supplementation in steers. © 2017 Blackwell Verlag GmbH.

Anticonvulsant effects of a triheptanoin diet in two mouse chronic seizure models

PubMed Central

Willis, Sarah; Stoll, James; Sweetman, Lawrence; Borges, Karin

2010-01-01

We hypothesized that in epileptic brains citric acid cycle intermediate levels may be deficient leading to hyperexcitability. Anaplerosis is the metabolic refilling of deficient metabolites. Our goal was to determine the anticonvulsant effects of feeding triheptanoin, the triglyceride of anaplerotic heptanoate. CF1 mice were fed 0-35% calories from triheptanoin. Body weights and dietary intake were similar in mice fed triheptanoin vs. standard diet. Triheptanoin feeding increased blood propionyl-carnitine levels, signifying its metabolism. 35%, but not 20%, triheptanoin delayed development of corneal kindled seizures. After pilocarpine-induced status epilepticus (SE), triheptanoin feeding increased the pentylenetetrazole tonic seizure threshold during the chronically epileptic stage. Mice in the chronically epileptic stage showed various changes in brain metabolite levels, including a reduction in malate. Triheptanoin feeding largely restored a reduction in propionyl-CoA levels and increased methylmalonyl-CoA levels in SE mice. In summary, triheptanoin was anticonvulsant in two chronic mouse models and increased levels of anaplerotic precursor metabolites in epileptic mouse brains. The mechanisms of triheptanoin's effects and its efficacy in humans suffering from epilepsy remain to be determined. PMID:20691264

The liberating effect of weight loss supplements on dietary control: a field experiment.

PubMed

Chang, Yevvon Y; Chiou, Wen-Bin

2014-09-01

Taking weight-loss supplements may create illusion of protection against weight gain and thereby loosen subsequent dietary self-control. The current study examined whether taking weight-loss supplement would increase food intake and further tested whether positive attitudes toward supplements would increase susceptibility to overeating. Participants were randomly assigned to take either a known placebo or a purported weight loss supplement (actually, the same placebo). After supplement provision, participants' actual food consumption at a reward buffet lunch was recorded. Compared with controls, participants receiving a purported weight loss supplement ate more food at the reward buffet. Perceived progress toward the goal of weight reduction mediated the connection between use of weight loss supplements and subsequent food consumption. Participants with more positive attitudes toward weight loss supplements were more susceptible to the liberating effect of taking weight loss supplements on food intake. Using weight loss supplements may produce unintended consequences on dietary self-regulation. The public should pay more attention to the notion of psychological liberation when using weight loss supplements. Copyright © 2014 Elsevier Inc. All rights reserved.

A simulation study on the constancy of cardiac energy metabolites during workload transition.

PubMed

Saito, Ryuta; Takeuchi, Ayako; Himeno, Yukiko; Inagaki, Nobuya; Matsuoka, Satoshi

2016-12-01

The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant during physiological cardiac workload transition. How this is accomplished is not yet clarified, though Ca 2+ has been suggested to be one of the possible mechanisms. We constructed a detailed mathematical model of cardiac mitochondria based on experimental data and studied whether known Ca 2+ -dependent regulation mechanisms play roles in the metabolite constancy. Model simulations revealed that the Ca 2+ -dependent regulation mechanisms have important roles under the in vitro condition of isolated mitochondria where malate and glutamate were mitochondrial substrates, while they have only a minor role and the composition of substrates has marked influence on the metabolite constancy during workload transition under the simulated in vivo condition where many substrates exist. These results help us understand the regulation mechanisms of cardiac energy metabolism during physiological cardiac workload transition. The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant over a wide range of cardiac workload, though the mechanisms are not yet clarified. One possible regulator of mitochondrial metabolism is Ca 2+ , because it activates several mitochondrial enzymes and transporters. Here we constructed a mathematical model of cardiac mitochondria, including oxidative phosphorylation, substrate metabolism and ion/substrate transporters, based on experimental data, and studied whether the Ca 2+ -dependent activation mechanisms play roles in metabolite constancy. Under the in vitro condition of isolated mitochondria, where malate and glutamate were used as mitochondrial substrates, the model well reproduced the Ca 2+ and inorganic phosphate (P i ) dependences of oxygen consumption, NADH level and mitochondrial membrane potential. The Ca 2+ -dependent activations of the aspartate/glutamate carrier and the F 1 F o -ATPase, and the P i -dependent activation of Complex III were key factors in reproducing the experimental data. When the mitochondrial model was implemented in a simple cardiac cell model, simulation of workload transition revealed that cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyt ) within the physiological range markedly increased NADH level. However, the addition of pyruvate or citrate attenuated the Ca 2+ dependence of NADH during the workload transition. Under the simulated in vivo condition where malate, glutamate, pyruvate, citrate and 2-oxoglutarate were used as mitochondrial substrates, the energy metabolites were more stable during the workload transition and NADH level was almost insensitive to [Ca 2+ ] cyt . It was revealed that mitochondrial substrates have a significant influence on metabolite constancy during cardiac workload transition, and Ca 2+ has only a minor role under physiological conditions. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

[Effect of jingui shenqi pill and its disassembled recipes on ovarian functions in shen yang deficiency female rats].

PubMed

Long, Yong-Ling; Li, Zheng-Mu

2013-07-01

To observe the effect of Jingui Shenqi Pill (JSP) and its disassembled recipes (supplementing Shen yang, supplementing Shen yin, and supplementing Shen yang and Shen yin) on ovarian functions of female rats of Shen yang deficiency syndrome (SYDS). Totally 55 SD female rats were randomly divided into 5 groups, i.e., the normal control group, the model group, the Shen yang supplementing group, the Shen yin supplementing group, the Shen yang and Shen yin supplementing group, 11 in each group. Except the normal control group, rats in the rest group were injected with hydrocortisone at the daily dose of 25 mg/kg at the muscle of femoribus internus for 12 successive days. From the 13th day after successful modeling, rats were administered by gastrogavage with different recipes at the dose of 1 mL/100 g (2.75 g/kg Shen yang supplementing recipe; 6.25 g/kg Shen yin supplementing recipe; 6.75 g/kg JSP), once daily for 20 successive days. Equal volume of normal saline was given to those in the normal control group and the model group, once daily for 20 successive days. Blood was withdrawn from the orbit on the 2nd day after intervention. The serum estradiol (E2) and progesterone (P) were detected using ELISA. The weight of uterus and ovarian index (VI) were calculated. The pathological changes were observed by HE staining. The general condition of rats in the Shen yang and Shen yin supplementing group were improved. The body weight (g) was added by 35.0 +/- 12.5 in the normal control group, 16.7 +/- 7.4 in the model group, 20.2 +/- 6.9 in the Shen yang supplementing group, 18.3 +/- 3.6 in the Shen yin supplementing group, and 29.4 +/- 12.2 in the Shen yang and Shen yin supplementing group. The uterus VI (mg/100 g) was 183.4 +/- 11.6 in the normal control group,144.0 +/- 6.5 in the model group,158.7 +/- 6.3 in the Shen yang supplementing group,152.1 +/- 6.9 in the Shen yin supplementing group, and 172.8 +/- 8.1 in the Shen yang and Shen yin supplementing group. The ovarian VI (mg/100 g) were 32.9 +/- 2.4 in the normal control group, 22.6 +/- 1.1 in the model group, 25.0 +/- 1.4 in the Shen yang supplementing group, 23.0 +/- 0.4 in the Shen yin supplementing group, and 31.4 +/- 3.3 in the Shen yang and Shen yin supplementing group. Compared with the model group, the body weight and ovarian VI increased in the Shen yang supplementing group and the Shen yang and Shen yin supplementing group (P < 0.05, P < 0.01). The uterus VI increased in each medicated group (P < 0.05, P < 0.01). Compared with the Shen yang supplementing group and the Shen yin supplementing group, all indices increased in the Shen yang and Shen yin supplementing group (P < 0.05, P < 0.01). The E2 and P levels increased in the Shen yang supplementing group and the Shen yang and Shen yin supplementing group (P < 0.05, P < 0.01). The content of E2 (pg/mL) was 22.1 +/- 9.4 in the normal control group, 9.8 +/- 3.0 in the model group, 11.3 +/- 2.2 in the Shen yang supplementing group, 10.5 +/- 0.8 in the Shen yin supplementing group, and 16.0 +/- 5.5 in the Shen yang and Shen yin supplementing group. The content of P (ng/mL) was 14.6 +/- 7.5 in the normal control group, 4.3 +/- 1.8 in the model group, 8.3 +/- 2.8 in the Shen yang supplementing group, 5.9 +/- 2.9 in the Shen yin supplementing group, and 9.5 +/- 3.4 in the Shen yang and Shen yin supplementing group. Compared with the Shen yang supplementing group and the Shen yin supplementing group, the E2 level increased in the Shen yang and Shen yin supplementing group (P < 0.05, P < 0.01). Compared with the Shen yin supplementing group, the P level increased in the Shen yang and Shen yin supplementing group (P < 0.05). Compared with the model group, the ovarian follicle at each stage increased and pathological follicular ovarian follicles decreased in the Shen yang and Shen yin supplementing group (P < 0.01). Less primary follicles, secondary follicles, and mature follicles could be seen in the Shen yang supplementing group and the Shen yin supplementing group. The total numbers of all-level follicles were obviously higher in the Shen yang and Shen yin supplementing group than in the Shen yang supplementing group and the Shen yin supplementing group (P < 0.05). The number of pathological follicles was obviously less in the Shen yang and Shen yin supplementing group than in the Shen yin supplementing group (P < 0.05). As for SYDS, JSP and its dissembled recipes could improve damaged ovarian functions to some degree. But better effect could not be obtained by Shen yang supplementing method or Shen yin supplementing method alone. Shen yang and Shen yin supplementing method could elevate the efficacy.

Effects of Almond- and Olive Oil-Based Docosahexaenoic- and Vitamin E-Enriched Beverage Dietary Supplementation on Inflammation Associated to Exercise and Age

PubMed Central

Capó, Xavier; Martorell, Miquel; Sureda, Antoni; Riera, Joan; Drobnic, Franchek; Tur, Josep Antoni; Pons, Antoni

2016-01-01

n-3-polyunsaturated fatty acids and polyphenols are potential key factors for the treatment and prevention of chronic inflammation associated to ageing and non-communicable diseases. The aim was to analyse effects of an almond and olive oil beverage enriched with α-tocopherol and docosahexaenoic, exercise and age on inflammatory plasma markers, and immune gene expression in peripheral blood mononuclear cells (PBMCs). Five young and five senior athletes who were supplemented for five weeks with a functional beverage performed a stress test under controlled conditions before and after beverage supplementation. Blood samples were taken immediately before and 1 h after each test. Plasma, erythrocytes and PBMCs were isolated. Beverage supplementation increased plasmatic Tumour Necrosis Factor α (TNFα) levels depending on age and exercise. Exercise increased plasma non esterified fatty acids (NEFAs), soluble Intercellular adhesion molecule 3 (sICAM3) and soluble L-selectin (sL-Selectin), and this increase was attenuated by the supplementation. Exercise increased PGE2 plasma levels in supplemented young and in senior placebo athletes. Exercise increased NFkβ-activated levels in PBMCs, which are primed to a pro-inflammatory response increasing pro-inflammatory genes expression after the exercise mainly in the young group after the supplementation. The functional beverage supplementation to young athletes enhances a pro-inflammatory circulating environment in response to the exercise that was less evident in the senior group. PMID:27735833

Effects of Almond- and Olive Oil-Based Docosahexaenoic- and Vitamin E-Enriched Beverage Dietary Supplementation on Inflammation Associated to Exercise and Age.

PubMed

Capó, Xavier; Martorell, Miquel; Sureda, Antoni; Riera, Joan; Drobnic, Franchek; Tur, Josep Antoni; Pons, Antoni

2016-10-09

n -3-polyunsaturated fatty acids and polyphenols are potential key factors for the treatment and prevention of chronic inflammation associated to ageing and non-communicable diseases. The aim was to analyse effects of an almond and olive oil beverage enriched with α-tocopherol and docosahexaenoic, exercise and age on inflammatory plasma markers, and immune gene expression in peripheral blood mononuclear cells (PBMCs). Five young and five senior athletes who were supplemented for five weeks with a functional beverage performed a stress test under controlled conditions before and after beverage supplementation. Blood samples were taken immediately before and 1 h after each test. Plasma, erythrocytes and PBMCs were isolated. Beverage supplementation increased plasmatic Tumour Necrosis Factor α (TNFα) levels depending on age and exercise. Exercise increased plasma non esterified fatty acids (NEFAs), soluble Intercellular adhesion molecule 3 (sICAM3) and soluble L-selectin (sL-Selectin), and this increase was attenuated by the supplementation. Exercise increased PGE2 plasma levels in supplemented young and in senior placebo athletes. Exercise increased NFkβ-activated levels in PBMCs, which are primed to a pro-inflammatory response increasing pro-inflammatory genes expression after the exercise mainly in the young group after the supplementation. The functional beverage supplementation to young athletes enhances a pro-inflammatory circulating environment in response to the exercise that was less evident in the senior group.

Effect of copper on performance, carcass characteristics, and muscle fatty acid composition of meat goat kids.

PubMed

Huang, Y L; Wang, Y; Spears, J W; Lin, X; Guo, C H

2013-10-01

An experiment was conducted to determine the effects of dietary Cu on performance, carcass characteristics, and muscle fatty acid composition in meat goats. Thirty five Jianyang Big-ear goat (JYB) kids (average BW 20.3 ± 0.6 kg and age 3 to 4 mo) were stratified by weight and randomly assigned to 1 of 7 experimental treatments (n = 5 goats per treatment). Treatments consisted of: 1) control (no supplemental Cu; 14.3 mg Cu/kg DM), 2) 20 mg supplemental Cu/kg DM, 3) 40 mg supplemental Cu/kg DM, 4) 80 mg supplemental Cu/kg DM, 5) 160 mg supplemental Cu/kg DM, 6) 320 mg supplemental Cu/kg DM, and 7) 640 mg supplemental Cu/kg DM. Copper was supplemented from CuSO4•5H2O (25.2% Cu). Goats were individually fed a concentrate-hay based diet for 96 d. Performance was not affected by Cu concentration. Liver Cu concentration was increased (P < 0.01) with Cu supplementation. Goats supplemented with 0 or 20 mg Cu/kg DM had lower (P < 0.01) liver Cu concentrations than the other treatments. Backfat depth (P < 0.01) and intramuscular fat (IMF) content (P < 0.01) were also increased with Cu supplementation. However, Cu-supplemented goats had lower (P = 0.04) longissimus muscle area (LMA) compared with control. Dietary Cu supplementation increased the percentage of C14:0 (P < 0.01), C20:4 (P < 0.01), and total polyunsaturated fatty acids (P = 0.03), decreased C18:1 trans (P = 0.04), and tended to decrease C18:0 (P = 0.08) in LM. Other fatty acids detected were not affected by dietary Cu supplementation (P > 0.10). These results indicate that JYB goats can tolerate up to 640 mg Cu/kg DM for 96 d without adverse effects on performance, but fat deposition and fatty acid composition in the body could be altered by Cu supplementation as low as 20 mg/kg of diet with high concentrate-hay. Copper supplementation increased backfat depth, IMF, and percentage of polyunsaturated fatty acids in LM and decreased LMA in the carcass of JYB goats.

Supplement use by Young Athletes

PubMed Central

McDowall, Jill Anne

2007-01-01

This paper reviews studies of supplement use among child and adolescent athletes, focusing on prevalence and type of supplement use, as well as gender comparisons. Supplement use among adult athletes has been well documented however there are a limited number of studies investigating supplement use by child and adolescent athletes. A trend in the current literature revealed that the most frequently used supplements are in the form of vitamin and minerals. While health and illness prevention are the main reasons for taking supplements, enhanced athletic performance was also reported as a strong motivating factor. Generally, females are found to use supplements more frequently and are associated with reasons of health, recovery, and replacing an inadequate diet. Males are more likely to report taking supplements for enhanced performance. Both genders equally rated increased energy as another reason for engaging in supplement use. Many dietary supplements are highly accessible to young athletes and they are particularly vulnerable to pressures from the media and the prospect of playing sport at increasingly elite levels. Future research should provide more direct evidence regarding any physiological side effects of taking supplements, as well as the exact vitamin and mineral requirements for child and adolescent athletes. Increased education for young athletes regarding supplement use, parents and coaches should to be targeted to help the athletes make the appropriate choices. Key pointsSupplement use among the child and adolescent athlete population is widespread with the most frequently used supplement being a form of vitamin/mineral supplement.The effects of supplement use on the growth and development of children and adolescents remain unclear and thus use of supplements by this population should be discouraged.It is likely that there is a misunderstanding as to the role of vitamins and minerals in the diet, their function in maintaining overall health, their role in athletic performance, and how they are best obtained from the diet therefore further education for adolescent athletes and athletes in general is needed. PMID:24149420

Dietary Crude Lecithin Increases Systemic Availability of Dietary Docosahexaenoic Acid with Combined Intake in Rats.

PubMed

van Wijk, Nick; Balvers, Martin; Cansev, Mehmet; Maher, Timothy J; Sijben, John W C; Broersen, Laus M

2016-07-01

Crude lecithin, a mixture of mainly phospholipids, potentially helps to increase the systemic availability of dietary omega-3 polyunsaturated fatty acids (n-3 PUFA), such as docosahexaenoic acid (DHA). Nevertheless, no clear data exist on the effects of prolonged combined dietary supplementation of DHA and lecithin on RBC and plasma PUFA levels. In the current experiments, levels of DHA and choline, two dietary ingredients that enhance neuronal membrane formation and function, were determined in plasma and red blood cells (RBC) from rats after dietary supplementation of DHA-containing oils with and without concomitant dietary supplementation of crude lecithin for 2-3 weeks. The aim was to provide experimental evidence for the hypothesized additive effects of dietary lecithin (not containing any DHA) on top of dietary DHA on PUFA levels in plasma and RBC. Dietary supplementation of DHA-containing oils, either as vegetable algae oil or as fish oil, increased DHA, eicosapentaenoic acid (EPA), and total n-3 PUFA, and decreased total omega-6 PUFA levels in plasma and RBC, while dietary lecithin supplementation alone did not affect these levels. However, combined dietary supplementation of DHA and lecithin increased the changes induced by DHA supplementation alone. Animals receiving a lecithin-containing diet also had a higher plasma free choline concentration as compared to controls. In conclusion, dietary DHA-containing oils and crude lecithin have synergistic effects on increasing plasma and RBC n-3 PUFA levels, including DHA and EPA. By increasing the systemic availability of dietary DHA, dietary lecithin may increase the efficacy of DHA supplementation when their intake is combined.

Creatine Supplementation Increases Total Body Water in Soccer Players: a Deuterium Oxide Dilution Study.

PubMed

Deminice, R; Rosa, F T; Pfrimer, K; Ferrioli, E; Jordao, A A; Freitas, E

2016-02-01

This study aimed to evaluate changes in total body water (TBW) in soccer athletes using a deuterium oxide dilution method and bioelectrical impedance (BIA) formulas after 7 days of creatine supplementation. In a double-blind controlled manner, 13 healthy (under-20) soccer players were divided randomly in 2 supplementation groups: Placebo (Pla, n=6) and creatine supplementation (CR, n=7). Before and after the supplementation period (0.3 g/kg/d during 7 days), TBW was determined by deuterium oxide dilution and BIA methods. 7 days of creatine supplementation lead to a large increase in TBW (2.3±1.0 L) determined by deuterium oxide dilution, and a small but significant increase in total body weight (1.0±0.4 kg) in Cr group compared to Pla. The Pla group did not experience any significant changes in TBW or body weight. Although 5 of 6 BIA equations were sensitive to determine TBW changes induced by creatine supplementation, the Kushner et al. 16 method presented the best concordance levels when compared to deuterium dilution method. In conclusion, 7-days of creatine supplementation increased TBW determined by deuterium oxide dilution or BIA formulas. BIA can be useful to determine TBW changes promoted by creatine supplementation in soccer athletes, with special concern for formula choice. © Georg Thieme Verlag KG Stuttgart · New York.

Leucine supplementation via drinking water reduces atherosclerotic lesions in apoE null mice

PubMed Central

Zhao, Yang; Dai, Xiao-yan; Zhou, Zhou; Zhao, Ge-xin; Wang, Xian; Xu, Ming-jiang

2016-01-01

Aim: Recent evidence suggests that the essential amino acid leucine may be involved in systemic cholesterol metabolism. In this study, we investigated the effects of leucine supplementation on the development of atherosclerosis in apoE null mice. Methods: ApoE null mice were fed with chow supplemented with leucine (1.5% w/v) in drinking water for 8 week. Aortic atherosclerotic lesions were examined using Oil Red O staining. Plasma lipoprotein-cholesterol levels were measured with fast protein liquid chromatography. Hepatic gene expression was detected using real-time PCR and Western blot analyses. Results: Leucine supplementation resulted in 57.6% reduction of aortic atherosclerotic lesion area in apoE null mice, accompanied by 41.2% decrease of serum LDL-C levels and 40.2% increase of serum HDL-C levels. The body weight, food intake and blood glucose level were not affected by leucine supplementation. Furthermore, leucine supplementation increased the expression of Abcg5 and Abcg8 (that were involved in hepatic cholesterol efflux) by 1.28- and 0.86-fold, respectively, and significantly increased their protein levels. Leucine supplementation also increased the expression of Srebf1, Scd1 and Pgc1b (that were involved in hepatic triglyceride metabolism) by 3.73-, 1.35- and 1.71-fold, respectively. Consequently, leucine supplementation resulted in 51.77% reduction of liver cholesterol content and 2.2-fold increase of liver triglyceride content. Additionally, leucine supplementation did not affect the serum levels of IL-6, IFN-γ, TNF-α, IL-10 and IL-12, but markedly decreased the serum level of MCP-1. Conclusion: Leucine supplementation effectively attenuates atherosclerosis in apoE null mice by improving the plasma lipid profile and reducing systemic inflammation. PMID:26687933

[USE, EFFECTS, AND KNOWLEDGE OF THE NUTRITIONAL SUPPLEMENTS FOR THE SPORT IN UNIVERSITY STUDENTS].

PubMed

Colls Garrido, Christian; Gómez-Urquiza, José Luis; Cañadas-De la Fuente, Guillermo Arturo; Fernández-Castillo, Rafael

2015-08-01

consumption and marketing of supplements that help improve athletic performance has increased in semi-professional sport. Moreover, in the market are increasingly a wide variety of such products pressure and high performance requirements push many young athletes to have recourse to the use of supplements to improve your fitness. However, this type of treatment should be advised and guided by an expert since improper use of such supplements favors the appearance of adverse effects and can be harmful to the health of the individual. to know the use of supplements to improve athletic performance by college athletes methods: was a systematic review in the Pubmed database, care, BIREME CUIDEN, BIREME (IBECS y Scielo) and CINHAL limited to articles published in the last ten years. 25 articles were analyzed. The main themes were found in the literature reviewed have been three: the "levels of supplements to increase athletic performance in college students", "effect of sports supplements" and "knowledge, behaviors and motivations for sports supplements". taking into account that the around 55% of University athletes using supplements but show a lack significant knowledge is necessary to provide a health education on such supplements. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

The ALMT family of organic acid transporters in plants and their involvement in detoxification and nutrient security

USDA-ARS?s Scientific Manuscript database

About a decade ago, members of a new protein family of anion channels were discovered on the basis of their ability to confer on plants the tolerance towards toxic aluminum ions in the soil. The efflux of Al3+ chelating malate anions through these channels is stimulated by external Al3+ ions. This f...

Temperature shift experiments suggest that metabolic impairment and enhanced rates of photorespiration decrease organic acid levels in soybean leaflets exposed to supra-optimal growth temperatures

USDA-ARS?s Scientific Manuscript database

Citrate, malate, malonate, fumarate and succinate in soybean leaflets decreased 40 to 80% when plants were grown continuously in controlled environment chambers at 36/28 compared to 28/20 °C. Glycerate was not temperature responsive in this study. Temperature effects on the above mentioned organi...

Reduction of α-oxo carboxyylic acids by pigeon liver `malic' enzyme

PubMed Central

Tang, Chung L.; Hsu, Robert Y.

1973-01-01

1. Pigeon liver `malic' enzyme [l-malate–NADP+ oxidoreductase (decarboxylating); EC 1.1.1.40] was shown to catalyse the reductase reaction: [Formula: see text] l-Malate was identified as the reaction product, and was formed in stoicheiometric amount. 2. In addition to oxaloacetate and pyruvate, a number of other α-oxo carboxylic acids were also reduced. PMID:4764261

Carbon Dioxide Metabolism in Leaf Epidermal Tissue 1

PubMed Central

Willmer, C. M.; Pallas, J. E.; Black, C. C.

1973-01-01

A number of plant species were surveyed to obtain pure leaf epidermal tissue in quantity. Commelina communis L. and Tulipa gesnariana L. (tulip) were chosen for further work. Chlorophyll a/b ratios of epidermal tissues were 2.41 and 2.45 for C. communis and tulip, respectively. Phosphoenolpyruvate carboxylase, ribulose-1,5-diphosphate carboxylase, malic enzyme, and NAD+ and NADP+ malate dehydrogenases were assayed with epidermal tissue and leaf tissue minus epidermal tissue. In both species, there was less ribulose 1,5-diphosphate than phosphoenolpyruvate carboxylase activity in epidermal tissue whether expressed on a protein or chlorophyll basis whereas the reverse was true for leaf tissue minus epidermal tissue. In both species, malic enzyme activities were higher in epidermal tissue than in the remaining leaf tissue when expressed on a protein or chlorophyll basis. In both species, NAD+ and NADP+ malate dehydrogenase activities were higher in the epidermal tissue when expressed on a chlorophyll basis; however, on a protein basis, the converse was true. Microautoradiography of C. communis epidermis and histochemical tests for keto acids suggested that CO2 fixation occurred predominantly in the guard cells. The significance and possible location of the enzymes are discussed in relation to guard cell metabolism. Images PMID:16658581

[Characterization of a malic enzyme isoform V from Mucor circinelloides].

PubMed

Zhang, Yingtong; Chen, Haiqin; Song, Yuanda; Zhang, Hao; Chen, Yongquan; Chen, Wei

2016-02-04

We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using. Ni-NTA column and characterized subsequently. The optimum conditions were pH at 8.0 and temperature at 33 degrees C. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K(m) for L-malate and NADP+ were 0.74960 ± 0.06120 mmol/L and 0.22070 ± 0.01810 mmol/L, the V(max) for L-malate and NADP+ were 72.820 ± 1.077 U/mg and 86.110 ± 1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn2+, Mg2+, Co2+, Ni2+ could activate BLME1, whereas Ca2+, Cu2+ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

PubMed Central

Hofhuis, Julia; Schueren, Fabian; Nötzel, Christopher; Lingner, Thomas; Gärtner, Jutta; Jahn, Olaf

2016-01-01

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases. PMID:27881739

Molecular mechanisms of long noncoding RNAs on gastric cancer

PubMed Central

Li, Tianwen; Mo, Xiaoyan; Fu, Liyun; Xiao, Bingxiu; Guo, Junming

2016-01-01

Long noncoding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Aberrant expression of lncRNAs has been found associated with gastric cancer, one of the most malignant tumors. By complementary base pairing with mRNAs or forming complexes with RNA binding proteins (RBPs), some lncRNAs including GHET1, MALAT1, and TINCR may mediate mRNA stability and splicing. Other lncRNAs, such as BC032469, GAPLINC, and HOTAIR, participate in the competing endogenous RNA (ceRNA) network. Under certain circumstances, ANRIL, GACAT3, H19, MEG3, and TUSC7 exhibit their biological roles by associating with microRNAs (miRNAs). By recruiting histone-modifying complexes, ANRIL, FENDRR, H19, HOTAIR, MALAT1, and PVT1 may inhibit the transcription of target genes in cis or trans. Through these mechanisms, lncRNAs form RNA-dsDNA triplex. CCAT1, GAPLINC, GAS5, H19, MEG3, and TUSC7 play oncogenic or tumor suppressor roles by correlated with tumor suppressor P53 or onco-protein c-Myc, respectively. In conclusion, interaction with DNA, RNA and proteins is involved in lncRNAs’ participation in gastric tumorigenesis and development. PMID:26788991

Molecular docking and molecular dynamics simulations of fumarate hydratase and its mutant H235N complexed with pyromellitic acid and citrate.

PubMed

Subasri, S; Chaudhary, Santosh Kumar; Sekar, K; Kesherwani, Manish; Velmurugan, D

2017-12-01

Fumarase catalyzes the reversible, stereospecific hydration/dehydration of fumarate to L-malate during the Kreb's cycle. In the crystal structure of the tetrameric fumarase, it was found that some of the active site residues S145, T147, N188 G364 and H235 had water-mediated hydrogen bonding interactions with pyromellitic acid and citrate which help to the protonation state for the conversion of fumarate to malate. When His 235 is mutated with Asn (H235N), water-mediated interactions were lost due to the shifting of active site water molecule by 0.7 Å away. Molecular dynamics (MD) simulations were also carried out by NAMD and analyzed using Assisted Model Building with Energy Refinement (AMBER) program to better understand the conformational stability and other aspects during the binding of pyromellitic acid and citrate with native and mutant FH. The role of hydrogen bonds and hydrophobic interactions was also analyzed. The present study confirms that the H235N mutation has a major effect on the catalytic activity of fumarase which is evident from the biochemical studies.

Improvement of constraint-based flux estimation during L-phenylalanine production with Escherichia coli using targeted knock-out mutants.

PubMed

Weiner, Michael; Tröndle, Julia; Albermann, Christoph; Sprenger, Georg A; Weuster-Botz, Dirk

2014-07-01

Fed-batch production of the aromatic amino acid L-phenylalanine was studied with recombinant Escherichia coli strains on a 15 L-scale using glycerol as carbon source. Flux Variability Analysis (FVA) was applied for intracellular flux estimation to obtain an insight into intracellular flux distribution during L-phenylalanine production. Variability analysis revealed great flux uncertainties in the central carbon metabolism, especially concerning malate consumption. Due to these results two recombinant strains were genetically engineered differing in the ability of malate degradation and anaplerotic reactions (E. coli FUS4.11 ΔmaeA pF81kan and E. coli FUS4.11 ΔmaeA ΔmaeB pF81kan). Applying these malic enzyme knock-out mutants in the standardized L-phenylalanine production process resulted in almost identical process performances (e.g., L-phenylalanine concentration, production rate and byproduct formation). This clearly highlighted great redundancies in central metabolism in E. coli. Uncertainties of intracellular flux estimations by constraint-based analyses during fed-batch production of L-phenylalanine were drastically reduced by application of the malic enzyme knock-out mutants. © 2014 Wiley Periodicals, Inc.

In Vitro Analysis of Metabolite Transport Proteins.

PubMed

Roell, Marc-Sven; Kuhnert, Franziska; Zamani-Nour, Shirin; Weber, Andreas P M

2017-01-01

The photorespiratory cycle is distributed over four cellular compartments, the chloroplast, peroxisomes, cytoplasm, and mitochondria. Shuttling of photorespiratory intermediates between these compartments is essential to maintain the function of photorespiration. Specific transport proteins mediate the transport across biological membranes and represent important components of the cellular metabolism. Although significant progress was made in the last years on identifying and characterizing new transport proteins, the overall picture of intracellular metabolite transporters is still rather incomplete. The photorespiratory cycle requires at least 25 transmembrane transport steps; however to date only plastidic glycolate/glycerate transporter and the accessory 2-oxoglutarate/malate and glutamate/malate transporters as well as the mitochondrial transporter BOU1 have been identified. The characterization of transport proteins and defining their substrates and kinetics are still major challenges.Here we present a detailed set of protocols for the in vitro characterization of transport proteins. We provide protocols for the isolation of recombinant transport protein expressed in E. coli or Saccharomyces cerevisiae and the extraction of total leaf membrane protein for in vitro analysis of transporter proteins. Further we explain the process of reconstituting transport proteins in artificial lipid vesicles and elucidate the details of transport assays.

Photosynthetic carbon fixation characteristics of fruiting structures of Brassica campestris L

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singal, H.R.; Sheoran, I.S.; Singh, R.

1987-04-01

Activities of key enzymes of the Calvin cycle and C/sub 4/ metabolism, rates of CO/sub 2/ fixation, and the initial products of photosynthetic /sup 14/CO/sub 2/ fixation were determined in the podwall, seed coat (fruiting structures), and the subtending leaf (leaf below a receme) of Brassica campestris L. cv Toria. Compared to activities of ribulose-1,5-bisphosphate carboxylase and other Calvin cycle enzymes, e.g. NADP-glyceraldehyde-3-phosphate-dehydrogenase and ribulose-5-phosphate kinase, the activities of phosphoenol pyruvate carboxylase and other enzymes of C/sub 4/ metabolism, viz. NADP-malate dehydrogenase, NADP-malic enzyme, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, were generally much higher in seed than in podwallmore » and leaf. Podwall and leaf were comparable to each other. Pulse-chase experiments showed that in seed the major product of /sup 14/CO/sub 2/ assimilation was malate (in short time), whereas in podwall and leaf, the label initially appeared in 3-PGA. With time, the label moved to sucrose. In contrast to legumes, Brassica pods were able to fix net CO/sub 2/ during light. However, respiratory losses were very high during the dark period.« less

Cardiovascular and Metabolic Consequences of Testosterone Supplements in Young and Old Male Spontaneously Hypertensive Rats: Implications for Testosterone Supplements in Men.

PubMed

Dalmasso, Carolina; Patil, Chetan N; Yanes Cardozo, Licy L; Romero, Damian G; Maranon, Rodrigo O

2017-10-17

The safety of testosterone supplements in men remains unclear. In the present study, we tested the hypothesis that in young and old male spontaneously hypertensive rats (SHR), long-term testosterone supplements increase blood pressure and that the mechanism is mediated in part by activation of the renin-angiotensin system. In untreated males, serum testosterone exhibited a sustained decrease after 5 months of age, reaching a nadir by 18 to 22 months of age. The reductions in serum testosterone were accompanied by an increase in body weight until very old age (18 months). Testosterone supplements were given for 6 weeks to young (12 weeks-YMSHR) and old (21-22 months-OMSHR) male SHR that increased serum testosterone by 2-fold in young males and by 4-fold in old males. Testosterone supplements decreased body weight, fat mass, lean mass, and plasma leptin, and increased plasma estradiol in YMSHR but had no effect in OMSHR. Mean arterial pressure (MAP) was significantly higher in OMSHR than in YMSHR and testosterone supplements decreased MAP in OMSHR, but significantly increased MAP in YMSHR. Enalapril, the angiotensin-converting enzyme inhibitor, reduced MAP in both control and testosterone-supplemented YMSHR, but had a greater effect on MAP in testosterone-treated rats, suggesting the mechanism responsible for the increase in MAP in YMSHR is mediated at least in part by activation of the renin-angiotensin system. Taken together with previous studies, these data suggest that testosterone supplements may have differential effects on men depending on age, cardiovascular and metabolic status, and dose and whether given long-term or short-term. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Effects of feeding a Moringa oleifera rachis and twig preparation to dairy cows on their milk production and fatty acid composition, and plasma antioxidants.

PubMed

Zhang, Tingting; Si, Bingwen; Deng, Kaidong; Tu, Yan; Zhou, Chaolong; Diao, Qiyu

2018-01-01

We determined how supplementing the diet of lactating, multiparous Holstein dairy cows with a preparation of Moringa oleifera rachises and twigs affected their milk production and quality and the levels of plasma antioxidants. We found that milk yield increased in cows receiving the 6% (w/w) moringa supplement compared with that of the control. Addition of the moringa supplement increased the concentration of milk fat and decreased the somatic cell count in the milk. However, protein, glucose and total solid and urea nitrogen concentrations in the milk were the same for all treatments. The concentration of glutathione peroxidase increased for cows fed the moringa supplement compared with the control. The percentages of total unsaturated fatty acids, mono-unsaturated fatty acids, and polyunsaturated fatty acids including n-3 polyunsaturated fatty acid increased in the milk of cows fed the moringa supplement compared with those of the controls. Addition of the moringa supplement into the diet of lactating multiparous cows improved milk production and health status and modified milk fatty acid profile positively. The results suggested that moringa supplement could be used as a diet supplement for producing high quality and healthier milk. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Effect of glutamine supplementation on splanchnic metabolism in lactating dairy cows.

PubMed

Doepel, L; Lobley, G E; Bernier, J F; Dubreuil, P; Lapierre, H

2007-09-01

The suggestion that glutamine (Gln) might become conditionally essential postpartum in dairy cows has been examined through increased postruminal supply of Gln. Net nutrient flux through the splanchnic tissues and mammary gland was measured in 7 multiparous Holstein cows receiving abomasal infusions of water or 300 g/d of Gln for 21 d in a crossover design. Milk yield increased significantly (by 3%) in response to Gln supplementation, but the 2.4% increase in milk protein yield was not statistically significant. Glutamine treatment had no effect on portal or hepatic venous blood flows. Net portal appearance of Gln and Glu was increased by Gln supplementation, accounting for 83% of the infused dose with, therefore, only limited amounts available to provide additional energy to fuel metabolism of the portal-drained viscera. The extra net portal appearance of Gln was offset, however, by a corresponding increase in hepatic removal such that net Gln splanchnic release was not different between treatments. Nonetheless, the Gln treatment resulted in a 43% increase in plasma Gln concentration. Infusions of Gln did not affect splanchnic flux of other nonessential amino acids or of essential amino acids. Glutamine supplementation increased plasma urea-N concentration and tended to increase net hepatic urea flux, with a numerical increase in liver hepatic O2 consumption. There were no effects on glucose in terms of plasma concentration, net portal appearance, net liver release, or postliver supply, suggesting that Gln supplementation had no sparing effect on glucose metabolism. Furthermore, mammary uptake of glucose and amino acids, including Gln, was not affected by Gln supplementation. In conclusion, this study did not support the hypothesis that supplemental Gln would reduce glucose utilization across the gut or increase liver gluconeogenesis or mammary glutamine uptake to increase milk protein synthesis.

Type of supplemented simple sugar, not merely calorie intake, determines adverse effects on metabolism and aortic function in female rats

PubMed Central

Sangüesa, Gemma; Shaligram, Sonali; Akther, Farjana; Roglans, Núria; Laguna, Juan C.; Rahimian, Roshanak

2017-01-01

High consumption of simple sugars causes adverse cardiometabolic effects. We investigated the mechanisms underlying the metabolic and vascular effects of glucose or fructose intake and determined whether these effects are exclusively related to increased calorie consumption. Female Sprague-Dawley rats were supplemented with 20% wt/vol glucose or fructose for 2 mo, and plasma analytes and aortic response to vasodilator and vasoconstrictor agents were determined. Expression of molecules associated with lipid metabolism, insulin signaling, and vascular response were evaluated in hepatic and/or aortic tissues. Caloric intake was increased in both sugar-supplemented groups vs. control and in glucose- vs. fructose-supplemented rats. Hepatic lipogenesis was induced in both groups. Plasma triglycerides were increased only in the fructose group, together with decreased expression of carnitine palmitoyltransferase-1A and increased microsomal triglyceride transfer protein expression in the liver. Plasma adiponectin and peroxisome proliferator-activated receptor (PPAR)-α expression was increased only by glucose supplementation. Insulin signaling in liver and aorta was impaired in both sugar-supplemented groups, but the effect was more pronounced in the fructose group. Fructose supplementation attenuated aortic relaxation response to a nitric oxide (NO) donor, whereas glucose potentiated it. Phenylephrine-induced maximal contractions were reduced in the glucose group, which could be related to increased endothelial NO synthase (eNOS) phosphorylation and subsequent elevated basal NO in the glucose group. In conclusion, despite higher caloric intake in glucose-supplemented rats, fructose caused worse metabolic and vascular responses. This may be because of the elevated adiponectin level and the subsequent enhancement of PPARα and eNOS phosphorylation in glucose-supplemented rats. NEW & NOTEWORTHY This is the first study comparing the effects of glucose and fructose consumption on metabolic factors and aortic function in female rats. Our results show that, although total caloric consumption was higher in glucose-supplemented rats, fructose ingestion had a greater impact in inducing metabolic and aortic dysfunction. PMID:27923787

Metabolic networks and bioenergetics of Aurantiochytrium sp. B-072 during storage lipid formation

PubMed Central

Chaisawang, Montri; Verduyn, Cornelis; Chauvatcharin, Somchai; Suphantharika, Manop

2012-01-01

Baffled shake flask cultivation of Aurantiochytrium sp. B-072 was carried out at in a glucose-monosodium glutamate mineral medium at different C/N-ratios (30–165) with glucose fixed at 90 g/L. With increasing C/N-ratio, a modest increase in lipid content (60 to 73 % w/w) was observed whereas fat-free biomass decreased but overall biomass showed little variation. FA-profiles were not affected to a large extent by C/N-ratio and absolute docosahexaenoic (DHA)-levels fell in narrow range (5–6 g/L). However at C/N > 64 a rapid decrease in lipid synthetic rate and/or incomplete glucose utilization occurred. Glucose and FA-fluxes based on fat-free biomass peaked at a C/N ratio of 56. This condition was chosen for calculation of the redox balance (NAD(P)H) and energy (ATP) requirement and to estimate the in vivo P/O ratio during the main period of fatty acid biosynthesis. Several models with different routes for NADPH, acetyl-CoA formation and re-oxidation of OAA formed via ATP-citrate lyase were considered as these influence the redox- and energy balance. As an example, using a commonly shown scheme whereby NADPH is supplied by a cytosolic “transhydrogenase cycle” (pyruvate-OAA-malate-pyruvate) and OAA formed by ATP-citrate lyase is recycled via import into the mitochondria as malate, the calculated NADPH-requirement amounted to 5.5 with an ATP-demand of 10.5 mmol/(g fat-free biomass x h) and an in vivo P/O-ratio (not including non-growth associated maintenance) of 1.6. The lowest ATP requirement is found when acetyl-CoA would be transported directly from the mitochondria to the cytosol by carnitine acetyltransferase. Assay of some enzymes critical for NADPH supply indicates that activity of glucose-6-phosphate dehydrogenase, the first enzyme in the HMP pathway, is far insufficient for the required NADPH-flux and malic enzyme must be a major source. Activity of the latter (ca. 300 mU/mg protein) far exceeds that in oleaginous fungi and yeast. PMID:24031944

Plant based dietary supplement increases urinary pH

PubMed Central

Berardi, John M; Logan, Alan C; Rao, A Venket

2008-01-01

Background Research has demonstrated that the net acid load of the typical Western diet has the potential to influence many aspects of human health, including osteoporosis risk/progression; obesity; cardiovascular disease risk/progression; and overall well-being. As urinary pH provides a reliable surrogate measure for dietary acid load, this study examined whether a plant-based dietary supplement, one marketed to increase alkalinity, impacts urinary pH as advertised. Methods Using pH test strips, the urinary pH of 34 healthy men and women (33.9 +/- 1.57 y, 79.3 +/- 3.1 kg) was measured for seven days to establish a baseline urinary pH without supplementation. After this initial baseline period, urinary pH was measured for an additional 14 days while participants ingested the plant-based nutritional supplement. At the end of the investigation, pH values at baseline and during the treatment period were compared to determine the efficacy of the supplement. Results Mean urinary pH statistically increased (p = 0.03) with the plant-based dietary supplement. Mean urinary pH was 6.07 +/- 0.04 during the baseline period and increased to 6.21 +/- 0.03 during the first week of treatment and to 6.27 +/- 0.06 during the second week of treatment. Conclusion Supplementation with a plant-based dietary product for at least seven days increases urinary pH, potentially increasing the alkalinity of the body. PMID:18990209

Effects of oral iron(III) hydroxide polymaltose complex supplementation on hemoglobin increase, cognitive function, affective behavior and scholastic performance of adolescents with varying iron status: a single centre prospective placebo controlled study.

PubMed

Devaki, Pallaki Baby; Chandra, Ranjit K; Geisser, Peter

2009-01-01

To assess the effects of iron supplementation on iron status, cognitive function, affective behavior and scholastic performance in adolescents with varying iron status. Adolescents of both sexes with varying iron status were allocated to four treatment groups by using inclusion criteria. Three of the four groups (iron deficient anemic, iron deficient and control supplement) received iron(III) hydroxide polymaltose complex (IPC, Maltofer) containing 100 mg of elemental iron 6 days a week for 8 months, while the fourth group (control placebo) was given a placebo. Hematological parameters, cognitive function, affective behavior and scholastic performance were assessed at baseline, 4 months and 8 months of supplementation. Cognitive and scholastic performance test scores for the three supplemented groups increased from baseline to 4 months and from 4 months to 8 months (with concomitant increases in hematological parameters), whereas no increase was observed in the placebo group. No increase was seen in affective behavior scores for any of the groups during or after supplementation. IPC supplementation for eight months yielded significant improvements in cognitive function and scholastic performance in Indian adolescents with and without iron deficiency and anemia.

Effects of Beetroot Juice Supplementation on Cardiorespiratory Endurance in Athletes. A Systematic Review

PubMed Central

Domínguez, Raúl; Cuenca, Eduardo; Maté-Muñoz, José Luis; García-Fernández, Pablo; Serra-Paya, Noemí; Estevan, María Carmen Lozano; Herreros, Pablo Veiga; Garnacho-Castaño, Manuel Vicente

2017-01-01

Athletes use nutritional supplementation to enhance the effects of training and achieve improvements in their athletic performance. Beetroot juice increases levels of nitric oxide (NO), which serves multiple functions related to increased blood flow, gas exchange, mitochondrial biogenesis and efficiency, and strengthening of muscle contraction. These biomarker improvements indicate that supplementation with beetroot juice could have ergogenic effects on cardiorespiratory endurance that would benefit athletic performance. The aim of this literature review was to determine the effects of beetroot juice supplementation and the combination of beetroot juice with other supplements on cardiorespiratory endurance in athletes. A keyword search of DialNet, MedLine, PubMed, Scopus and Web of Science databases covered publications from 2010 to 2016. After excluding reviews/meta-analyses, animal studies, inaccessible full-text, and studies that did not supplement with beetroot juice and adequately assess cardiorespiratory endurance, 23 articles were selected for analysis. The available results suggest that supplementation with beetroot juice can improve cardiorespiratory endurance in athletes by increasing efficiency, which improves performance at various distances, increases time to exhaustion at submaximal intensities, and may improve the cardiorespiratory performance at anaerobic threshold intensities and maximum oxygen uptake (VO2max). Although the literature shows contradictory data, the findings of other studies lead us to hypothesize that supplementing with beetroot juice could mitigate the ergolytic effects of hypoxia on cardiorespiratory endurance in athletes. It cannot be stated that the combination of beetroot juice with other supplements has a positive or negative effect on cardiorespiratory endurance, but it is possible that the effects of supplementation with beetroot juice can be undermined by interaction with other supplements such as caffeine. PMID:28067808

Dietary supplementation with soybean lecithin increases pulmonary PAF bioactivity in asthmatic rats.

PubMed

Muehlmann, Luis A; Zanatta, Ana L; Farias, Carolina L A; Bieberbach, Eloyse W; Mazzonetto, Ana C; Michellotto, Pedro V; Fernandes, Luiz C; Nishiyama, Anita

2010-06-01

The prevalence of asthma has risen over the last few decades, and some studies correlate this with the greater consumption of polyunsaturated fatty acids (PUFAs). Dietary PUFAs are known to increase the susceptibility of biological structures to lipid peroxidation, a process by which platelet-activating factor (PAF)-like lipids can be generated. These lipids functionally mimic the bioactivity of PAF, a potent proinflammatory mediator that exerts several deleterious effects on asthma. Thus, this work aimed to investigate if dietary supplementation with soybean lecithin (SL), a source of PUFAs, increases lipid peroxidation and PAF bioactivity in lungs of asthmatic Wistar rats. Animals were separated into groups: control, supplemented, asthmatic, asthmatic supplemented with SL (2 g/kg body weight), asthmatic supplemented with SL (2 g/kg body weight) and DL-alpha-tocopheryl acetate (100 mg/kg body weight). Asthmatic inflammation increased pulmonary lipid peroxidation, PAF bioactivity, alveolar-capillary barrier permeability and production of nitric oxide. In asthmatics, dietary supplementation with SL promoted an increase in pulmonary lipid peroxidation and PAF bioactivity, and an increase in the permeability of the alveolar-capillary barrier. Moreover, the treatment of asthmatic rats with DL-alpha-tocopheryl acetate inhibited the lipid peroxidation and decreased the PAF bioactivity. Therefore, the increase in pulmonary PAF bioactivity in asthmatic individuals elicited by the dietary supplementation with SL probably involves the generation of PAF-like lipids. This finding suggests that PAF-like lipids may account for the deleterious effects of dietary PUFAs on asthma. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Intake, digestibility, and nitrogen retention by sheep supplemented with warm-season legume haylages or soybean meal.

PubMed

Foster, J L; Adesogan, A T; Carter, J N; Blount, A R; Myer, R O; Phatak, S C

2009-09-01

The high cost of commercial supplements necessitates evaluation of alternatives for ruminant livestock fed poor quality warm-season grasses. This study determined how supplementing bahiagrass haylage (Paspalum notatum Flügge cv. Tifton 9) with soybean [Glycine max (L.) Merr.] meal or warm-season legume haylages affected the performance of lambs. Forty-two Dorper x Katadhin lambs (27.5 +/- 5 kg) were fed for ad libitum intake of bahiagrass haylage (67.8% NDF, 9.6% CP) alone (control) or supplemented with soybean meal (18.8% NDF, 51.4% CP) or haylages of annual peanut [Arachis hypogaea (L.) cv. Florida MDR98; 39.6% NDF, 18.7% CP], cowpea [Vigna unguiculata (L.) Walp. cv. Iron clay; 44.1% NDF, 16.0% CP], perennial peanut (Arachis glabrata Benth. cv. Florigraze; 40.0% NDF, 15.8% CP), or pigeonpea [Cajanus cajan (L.) Millsp. cv. GA-2; 65.0% NDF, 13.7% CP]. Haylages were harvested at the optimal maturity for maximizing yield and nutritive value, wilted to 45% DM, baled, wrapped in polyethylene plastic, and ensiled for 180 d. Legumes were fed at 50% of the dietary DM, and soybean meal was fed at 8% of the dietary DM to match the average CP concentration (12.8%) of legume haylage-supplemented diets. Lambs were fed each diet for a 14-d adaptation period and a 7-d data collection period. Each diet was fed to 7 lambs in period 1 and 4 lambs in period 2. Pigeonpea haylage supplementation decreased (P < 0.01) DM and OM intake and digestibility vs. controls. Other legume haylages increased (P < 0.05) DM and OM intake vs. controls; however, only soybean meal supplementation increased (P = 0.01) DM digestibility. All supplements decreased (P = 0.05) NDF digestibility. Except for pigeonpea haylage, all supplements increased (P < 0.01) N intake, digestibility, and retention, and the responses were greatest (P = 0.04) with soybean meal supplementation. Microbial N synthesis was reduced (P = 0.02) by pigeonpea haylage supplementation, but unaffected (P = 0.05) by other supplements. Efficiency of microbial protein synthesis was unaffected (P = 0.05) by diet. Ruminal ammonia concentration was increased (P = 0.01) by all supplements, but only soybean meal and annual peanut haylage increased (P < 0.03) plasma urea-N concentrations. Perennial peanut, annual peanut, and cowpea haylages are promising protein supplements for growing lambs.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Alterations in uteroplacental hemodynamics during melatonin supplementation in sheep and cattle.

PubMed

Lemley, C O; Vonnahme, K A

2017-05-01

Compromised placental function can result in fetal growth restriction which is associated with greater risk of neonatal morbidity and mortality. Large increases in transplacental nutrient and waste exchange, which support the exponential increase in fetal growth during the last half of gestation, are dependent primarily on the rapid growth and vascularization of the uteroplacenta. The amplitude of melatonin secretion has been associated with improved oxidative status and altered cardiovascular function in several mammalian species; however, melatonin mediated alterations of uteroplacental capacity in sheep and cattle are lacking. Therefore, our laboratories are examining uteroplacental blood flow and fetal development during maternal melatonin supplementation. Using a mid- to late-gestation ovine model of intrauterine growth restriction, we examined uteroplacental blood flow and fetal growth during supplementation with 5 mg/d of dietary melatonin. Maternal nutrient restriction decreased uterine arterial blood flow, while melatonin supplementation increased umbilical arterial blood flow compared with non-supplemented controls. Although melatonin treatment did not rescue fetal weight in nutrient restricted ewes; we observed disproportionate fetal size and fetal organ development. Elevated fetal concentrations of melatonin may result in altered blood flow distribution during important time points of development. These melatonin specific responses on umbilical arterial hemodynamics and fetal development may be partially mediated through vascular melatonin receptors. Recently, we examined the effects of supplementing Holstein heifers with 20 mg/d of dietary melatonin during the last third of gestation. Uterine arterial blood flow was increased by 25% and total serum antioxidant capacity was increased by 43% in melatonin supplemented heifers vs. non-supplemented controls. In addition, peripheral concentrations of progesterone were decreased in melatonin supplemented heifers vs. non-supplemented controls. Using an in vitro model, melatonin treatment increased the activity of cytochrome P450 2C, a progesterone inactivating enzyme, which was blocked by treatment with the melatonin receptor antagonist, luzindole. Elucidating the consequences of specific hormonal supplements on the continual plasticity of placental function will allow us to determine important endogenous mediators of offspring growth and development.

Supplementation with sunflower seeds in beef cattle did not impact on oocyte and in vitro embryo production.

PubMed

Baltazar, A L; de Mattos, G M; Ropelli, B M; Firetti, Smg; Castilho, C; Pugliesi, G; Maldonado, Mbc; Binelli, M; Silva, Jof; Lupatini, G C; Lafuente, B S; Membrive, Cmb

2018-06-01

Supplementation with compounds rich in linoleic acid, including sunflower seed supplementation, promotes increase in conception rates in cows. We aimed to evaluate whether the sunflower seed (linoleic acid source) supplementation in beef donor females alters the plasma concentrations of cholesterol, triglycerides, HDL and LDL, increases the number and quality of oocytes, increases the cleavage rates and determines an improvement in number and quality of in vitro produced blastocysts. Thus, Nelore females were divided into two groups of 15 animals to receive supplementation with or without sunflower seed for 57 days. Females underwent follicular aspiration and the oocytes were subjected to in vitro embryo production. There was no difference (p > .1) between control group and group supplemented with sunflower seed on the number of displayed follicles; number of aspired oocytes; recovery rate; cleavage rate; number of embryos; number of blastocysts; embryos number of grades I and II; plasma concentrations of total cholesterol, triglycerides; HDL and LDL. Therefore, sunflower seed supplementation in oocyte donors did not increase the number and quality of oocytes, cleavage rates and the number and quality of blastocysts produced in vitro. © 2018 Blackwell Verlag GmbH.

Randomized Control Trial Assessing Impact of Increased Sunlight Exposure versus Vitamin D Supplementation on Lipid Profile in Indian Vitamin D Deficient Men

PubMed Central

Patwardhan, Vivek G.; Mughal, Zulf M.; Padidela, Raja; Chiplonkar, Shashi A.; Khadilkar, Vaman V.; Khadilkar, Anuradha V.

2017-01-01

Background: Despite abundance of sunshine in India, Vitamin D deficiency is common and therefore there is an increasing trend toward taking Vitamin D supplements either as prescription medicine or as a nutritional supplement. Studies have suggested that duration of sun exposure may influence serum lipid profile. Objectives: To study the effect of increased sunlight exposure versus Vitamin D supplementation on Vitamin D status and lipid profile in individuals with Vitamin D deficiency (25-hydroxyvitamin-D [25OHD] <50 nmol/L). Design: A prospective, randomized open-label trial was carried out in apparently healthy Indian men (40–60 years). Based on 25OHD concentrations, individuals were divided into control (>50 nmol/L, n = 50) and intervention (<50 nmol/L, n = 100) groups. Individuals from intervention group were randomly allocated to two groups; either “increased sunlight exposure group” (n = 50, received at least 20 min sunlight exposure to forearms and face between 11 a.m. and 3 p.m. over and above their current exposure) or “cholecalciferol supplement group” (n = 50, received oral cholecalciferol 1000 IU/day). Results: Significant increase in 25OHD concentrations was seen in both intervention groups (P < 0.01). Significant decrease in total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C), and low-density-lipoprotein cholesterol (LDL-C) was seen in individuals with increased sunlight exposure (P < 0.05). Cholecalciferol supplement group showed a significant increase in TC and HDL-C (P < 0.05) and insignificant increase in LDL-C. Conclusions: Increase in Vitamin D concentrations through sunlight exposure significantly reduced TC, LDL-C, and HDL-C concentrations, and cholecalciferol supplementation increased TC and HDL-C concentrations. PMID:28553593

The effects of increasing supplementation of zinc-amino acid complex on growth performance, carcass characteristics, and inflammatory response of beef cattle fed ractopamine hydrochloride.

PubMed

Genther-Schroeder, O N; Branine, M E; Hansen, S L

2016-08-01

Forty-two Angus crossbred steers (380 ± 5.3 kg) were enrolled in a finishing study to evaluate the influence of a supplemental Zn amino-acid complex (ZnAA; Availa-Zn) on performance and carcass characteristics of finishing steers in combination with ractopamine hydrochloride (RAC). Steers were stratified by BW into 7 pens of 6 steers each, and individual feed intake was measured. Steers were assigned to 1 of 4 treatments for 86 d (pre-RAC period): a dry-rolled corn-based diet supplemented with 60 mg Zn/kg DM from ZnSO and no supplemental ZnAA (CON; analyzed 88 mg Zn/kg DM; = 6) or CON diet supplemented with 30 (Zn30; = 12), 60 (Zn60; = 12), or 90 (Zn90; = 11) mg Zn/kg DM from ZnAA. Day 86 BW and G:F displayed a quadratic tendency ( = 0.09) with Zn60 steers being greater than the other treatments. Plasma cyclic adenosine monophosphate tended to linearly increase with increasing ZnAA ( = 0.10). On d 88, 6 of 12 steers (one of the 2 pens) receiving supplemental ZnAA was randomly selected to be supplemented with RAC at 300 mg∙steer∙d for the final 28 d of the experiment (RAC period). This created 7 final treatments: CON: no supplemental ZnAA, no RAC ( = 5); Zn30: Zn30, no RAC ( = 5); Zn30R: Zn30 + RAC ( = 6); Zn60: Zn60, no RAC ( = 6); Zn60R: Zn60 + RAC ( = 6); Zn90: Zn90, no RAC ( = 5); and Zn90R: Zn90 + RAC ( = 6). During the RAC period, as supplemental ZnAA increased within RAC-supplemented treatments, there was a linear increase in final BW, ADG, and G:F ( < 0.05). However, there was no effect of supplemental ZnAA on BW, ADG, or G:F during this period in non-RAC fed steers ( ≥ 0.44). Day 111 plasma Cu was increased, plasma Fe decreased, and leukocyte counts and serum interleukin-8 concentrations were greater ( < 0.05) in RAC-fed steers suggesting that RAC may elicit a mild inflammatory response. There was a tendency for increasing Zn supplementation to decrease plasma haptoglobin within RAC-fed steers ( = 0.07), suggesting that Zn may alter the inflammatory response. Overall, Zn60 improved growth performance during the pre-RAC period. Zinc supplemented as ZnAA appears to improve growth in combination with RAC supplementation, suggesting that Zn may enhance or support the biological function of RAC. Additionally, these results indicate that feeding RAC impacts trace mineral status, and potentially causes a non-specific inflammatory response, but further research is required to define this response.

Light-enhanced dark respiration in leaves, isolated cells and protoplasts of various types of C4 plants.

PubMed

Parys, Eugeniusz; Jastrzebski, Hubert

2006-04-01

The rate of respiratory CO2 evolution from the leaves of Zea mays, Panicum miliaceum, and Panicum maximum, representing NADP-ME, NAD-ME, and PEP-CK types of C4 plants, respectively, was increased by approximately two to four times after a period of photosynthesis. This light-enhanced dark respiration (LEDR) was a function of net photosynthetic rate specific to plant species, and was depressed by 1% O2. When malate, aspartate, oxaloacetate or glycine solution at 50 mM concentration was introduced into the leaves instead of water, the rate of LEDR was enhanced, far less in Z. mays (by 10-25%) than in P. miliaceum (by 25-35%) or P. maximum (by 40-75%). The enhancement of LEDR under glycine was relatively stable over a period of 1 h, whereas the remaining metabolites caused its decrease following a transient increase. The metabolites reduced the net photosynthesis rate in the two Panicum species, but not in Z. mays, where this process was stimulated by glycine. The bundle sheath cells from P. miliaceum exhibited a higher rate of LEDR than those of Z. mays and P. maximum. Glycine had no effect on the respiration rate of the cells, but malate increased in cells of Z. mays and P. miliaceum by about 50% and 30%, respectively. With the exception of aspartate, which stimulated both the O2 evolution and O2 uptake in P. maximum, the remaining metabolites reduced photosynthetic O2 evolution from bundle sheath cells in Panicun species. The net O2 exchange in illuminated cells of Z. mays did not respond to CO2 or metabolites. Leaf mesophyll protoplasts of Z. mays and P. miliaceum, and bundle sheath protoplasts of Z. mays, which are unable to fix CO2 photosynthetically, also produced LEDR, but the mesophyll protoplasts, compared with bundle sheath protoplasts, required twice the time of illumination to obtain the maximal rate. The results suggest that the substrates for LEDR in C4 plants are generated during a period of illumination not only via the Calvin cycle reactions, but also by the conversion of endogenous compounds present in leaf cells. The stimulation of LEDR under glycine is discussed in relation to its direct or indirect effect on mitochondrial respiration.

N-acetylcysteine supplementation controls total antioxidant capacity, creatine kinase, lactate, and tumor necrotic factor-alpha against oxidative stress induced by graded exercise in sedentary men.

PubMed

Leelarungrayub, Donrawee; Khansuwan, Raphiphat; Pothongsunun, Prapas; Klaphajone, Jakkrit

2011-01-01

Aim of this study was to evaluate the effects of short-term (7 days) N-acetylcysteine (NAC) at 1,200 mg daily supplementation on muscle fatigue, maximal oxygen uptake (VO(2max)), total antioxidant capacity (TAC), lactate, creatine kinase (CK), and tumor necrotic factor-alpha (TNF-α). Twenty-nine sedentary men (13 controls; 16 in the supplement group) from a randomized control were included. At before and after supplementation, fatigue index (FI) was evaluated in the quadriceps muscle, and performed a graded exercise treadmill test to induce oxidative stress, and as a measure of VO(2max). Blood samples were taken before exercise and 20 minutes after it at before and after supplementation, to determine TAC, CK, lactate, and TNF-α levels. Results showed that FI and VO(2max) increased significantly in the supplement group. After exercise decreased the levels of TAC and increased lactate, CK, and TNF-α of both groups at before supplementation. After supplementation, lactate, CK, and TNF-α levels significantly increased and TAC decreased after exercise in the control group. Whereas the TAC and lactate levels did not change significantly, but CK and TNF-α increased significantly in the supplement group. Therefore, this results showed that NAC improved the muscle fatigue, VO(2max), maintained TAC, controlled lactate production, but had no influence on CK and TNF-α.

Depressed antioxidant status in pregnant women on iron supplements: pathologic and clinical correlates.

PubMed

Anetor, J I; Ajose, O A; Adeleke, F N; Olaniyan-Taylor, G O; Fasola, F A

2010-08-01

Iron (Fe) remains a commonly prescribed supplement in pregnancy. Its possible pathologic potential is either uncommonly considered or ignored. We determined the antioxidant status in pregnant women with and without Fe supplements. Fifty-eight apparently healthy pregnant women on Fe supplements were selected for the study from the antenatal clinic of the University College Hospital, Ibadan, Nigeria. Fifty-five aged matched pregnant women who were not on Fe from various parishes of the Christ Apostolic Church, Ibadan (non-drug using Christian sect) were randomly selected as controls. Both groups were classified according to the trimesters of pregnancy. The gestational age in both pregnant women on Fe supplements and non-supplement pregnant women was similar. Fruit and vegetables consumption was higher in the supplement than in the non-supplement group (57.2% vs. 37.3%). Anthropometric indices, weight, height, and BMI, were also similar. But while the weight of the Fe supplement group decreased by nearly 3% in the third trimester, it increased by over 10% (p < 0.00) in the non-supplement group in the same period. Serum Fe level was significantly higher in the supplement than the non-supplement group (p < 0.001). In contrast, the levels of the antioxidants, ascorbic acid, copper (Cu), zinc (Zn), and bilirubin were all significantly decreased (p < 0.05, p < 0.001, p < 0.05, and p < 0.05, respectively). Uric acid level though also lower in the supplement group did not reach statistical significance (p > 0.05), while vitamin E was similar in both groups. There was relative stability of all antioxidants except uric acid, which declined from the first to the last trimester in the non-supplement group. The significantly higher Fe level in the second trimester was sustained in the third trimester though to a lesser degree (p < 0.05) and associated with significant decreases in the following antioxidant levels in the supplement group, ascorbic acid, bilirubin, Cu, and Zn (p < 0.02, p < 0.02, p < 0.02, and p < 0.001, respectively). Uric acid and vitamin E though lower in the supplement group were not significantly different. Remarkably, percentage changes between the first and third trimesters revealed that serum Fe increased by over 116% in the Fe supplement group, while it only increased by over 50% in the non-supplement group. This was associated with 23.50% decrease in ascorbate level (p < 0.003) in the supplement group, while it decreased by only 3.70% in the non-supplement group (p > 0.05). Again vitamin E decreased by 17.22% in the supplement group, while it decreased by only 7.30% in the non-supplement group during the period. Uric acid and bilirubin levels decreased by similar proportions during the period, while Zn decreased by 18.55% in the supplement group and by 14.86% in the non-supplement group. In contrast Cu increased by 7.20% in the supplement group, while it increased by only 2.96 in the non-supplement group. Additionally, all the antioxidants in the supplement group except vitamin E, viz, ascorbic acid, bilirubin, Cu, uric acid, and Zn, were significantly inversely correlated with serum Fe level (r - 0.299, p < 0.05, r - 0.278, p < 0.05, r - 0.383, p < 0.05, and r - 0.0369, p < 0.05). These data imply markedly depressed antioxidant status in the Fe supplement pregnant group with attendant oxidative stress (most probably pro-oxidant Fe-induced). This is associated with molecular and cellular damage as well as a number of pathologic and clinical correlates that underlie the exacerbation of morbidity and mortality in maternal and child populations, particularly in the developing countries. This appears to call for serious caution and prior evaluation of antioxidant and Fe status and during the use of Fe supplements in pregnancy for monitoring and prognostic purposes and to avert or ameliorate oxidative stress-induced pathologies in maternal and fetal systems.

Understanding supplement use: an application of temporal self-regulation theory.

PubMed

Allom, Vanessa; Mullan, Barbara; Clifford, Allison; Rebar, Amanda

2018-02-01

Vitamin and mineral supplement use is increasing worldwide. As evidence of the effects of these practices are equivocal at best, it is important to determine why individuals engage in vitamin and mineral supplement use through the application of a theoretical framework. The aim of this study was to employ temporal self-regulation theory (TST) to establish the determinants of supplement use. A cross-sectional design was used with a sample of 121 individuals who consume vitamins or minerals as dietary supplements. Hierarchical regression analyses were conducted to determine the influence of intention, habit, self-control, and the interaction between intention and habit and self-control on the frequency of supplement use. The model accounted for a significant proportion of variance in behaviour (R 2  = .44) with intention and habit significantly related to supplement use. Self-control moderated the relationship between intention and behaviour indicating that intention was not related to behaviour when self-control was low. The TST model offers a satisfactory account of supplement use and provides avenues for interventions aimed at increasing safe and beneficial supplement use and decreasing potentially harmful supplement use.

[Effects of a supplementation on sodium chloride or ammonium chloride on urolithic potential in the rabbit].

PubMed

Rückert, Cornelia; Siener, Roswitha; Ganter, Martin; Coenen, Manfred; Vervuert, Ingrid

2016-08-17

Reduction of urolithic potential by means of increased water intake and urine dilution through supplementation of sodium chloride (NaCl) or decrease of urine pH by supplementation of ammonium chloride (NH4Cl) in rabbits. Sixteen female, 6-month-old dwarf rabbits received the following three feeding regimens in a random order: complete feed without supplements = control; complete feed + 10 g NaCl/kg feed = NaCl; complete feed + 2.5 g NH4Cl/kg feed = NH4Cl. The diets were fed ad libitum over a period of 27 days without roughage. Water was provided ad libitum by a drinker. A 14-day wash-out-period (hay feeding) was performed between the different diets. Blood, faeces, and urine were collected at the beginning of each feeding period, after 21-day adaptation to the respective diet, and after the 3-day collection period. The following parameters were analysed: water and food intake as well as acid-base balance and mineral content in blood, urine, and faeces. NaCl supplementation numerically increased the daily water intake from 40.5 ± 14.4 ml/kg body weight (BW) (control) up to 49.5 ± 14.3 ml/kg BW and significantly increased the daily urine volume from 16.9 ± 7.8 ml/kg BW (control group) to 21.1 ± 7.4 ml/kg BW. The specific gravity of urine samples from NaCl supplementation decreased from 1.060 ± 0.008 to 1.044 ± 0.008. NH4Cl supplementation did not induce significant changes in urine pH, blood acid-base parameters, or calcium retention. Relative supersaturations (RSS) for calcium oxalate and calcium phosphate showed no significant changes after treatment. RSS for struvite increased from 360 ± 735 (after hay feeding) to 3312 ± 6188 on control feeding, 2910 ± 4913 with NaCl supplementation, and 3022 ± 6635 with NH4Cl supplementation (p < 0.05). NaCl supplementation significantly increased the urine volume and decreased its specific gravity. Therefore, NaCl supplementation might be an additional dietary treatment to increase the elimination of urine crystals in rabbits. NH4Cl supplementation did not induce acidification of the urine.

Lifelong alpha-tocopherol supplementation increases the median life span of C57BL/6 mice in the cold but has only minor effects on oxidative damage.

PubMed

Selman, Colin; McLaren, Jane S; Mayer, Claus; Duncan, Jackie S; Collins, Andrew R; Duthie, Garry G; Redman, Paula; Speakman, John R

2008-02-01

The effects of dietary antioxidant supplementation on oxidative stress and life span are confused. We maintained C57BL/6 mice at 7 +/- 2 degrees C and supplemented their diet with alpha-tocopherol from 4 months of age. Supplementation significantly increased (p = 0.042) median life span by 15% (785 days, n = 44) relative to unsupplemented controls (682 days, n = 43) and also increased maximum life span (oldest 10%, p = 0.028). No sex or sex by treatment interaction effects were observed on life span, with treatment having no effect on resting or daily metabolic rate. Lymphocyte and hepatocyte oxidative DNA damage and hepatic lipid peroxidation were unaffected by supplementation, but hepatic oxidative DNA damage increased with age. Using a cDNA macroarray, genes associated with xenobiotic metabolism were significantly upregulated in the livers of female mice at 6 months of age (2 months supplementation). At 22 months of age (18 months supplementation) this response had largely abated, but various genes linked to the p21 signaling pathway were upregulated at this time. We suggest that alpha-tocopherol may initially be metabolized as a xenobiotic, potentially explaining why previous studies observe a life span extension generally when lifelong supplementation is initiated early in life. The absence of any significant effect on oxidative damage suggests that the life span extension observed was not mediated via any antioxidant properties of alpha-tocopherol. We propose that the life span extension observed following alpha-tocopherol supplementation may be mediated via upregulation of cytochrome p450 genes after 2 months of supplementation and/or upregulation of p21 signaling genes after 18 months of supplementation. However, these signaling pathways now require further investigation to establish their exact role in life span extension following alpha-tocopherol supplementation.

Lipid oxidation in minced beef meat with added Krebs cycle substrates to stabilise colour.

PubMed

Yi, G; Grabež, V; Bjelanovic, M; Slinde, E; Olsen, K; Langsrud, O; Phung, V T; Haug, A; Oostindjer, M; Egelandsdal, B

2015-11-15

Krebs cycle substrates (KCS) can stabilise the colour of packaged meat by oxygen reduction. This study tested whether this reduction releases reactive oxygen species that may lead to lipid oxidation in minced meat under two different storage conditions. KCS combinations of succinate and glutamate increased peroxide forming potential (PFP, 1.18-1.32 mmol peroxides/kg mince) and thiobarbituric acid reactive substances (TBARS, 0.30-0.38 mg malondialdehyde (MDA) equivalents/kg mince) under low oxygen storage conditions. Both succinate and glutamate were metabolised. Moreover, under high oxygen (75%) storage conditions, KCS combinations of glutamate, citrate and malate increased PFP (from 1.22 to 1.29 mmol peroxides/kg) and TBARS (from 0.37 to 0.40 mg MDA equivalents/kg mince). Only glutamate was metabolised. The KCS combinations that were added to stabilise colour were metabolised during storage, and acted as pro-oxidants that promoted lipid oxidation in both high and low oxygen conditions. Copyright © 2015. Published by Elsevier Ltd.

Pre-anthesis CPPU low dosage application increases 'Hayward' kiwifruit weight without affecting the other qualitative and nutritional characteristics.

PubMed

Cruz-Castillo, J G; Baldicchi, A; Frioni, T; Marocchi, F; Moscatello, S; Proietti, S; Battistelli, A; Famiani, F

2014-09-01

In 2008, in Central Italy, a low dosage of CPPU solution, 4 μL L(-1) (6 hL/ha), was sprayed on the canopy of vines of 'Hayward' kiwifruit, at the "break of sepals", about one week before anthesis, to study its effects on fruit weight/size and on qualitative and nutritional characteristics. At harvest, CPPU, with respect to control, significantly increased the fresh weight by about 12% (+12.6 g fruit(-1)) and consequently the yield per vine, without affecting fruit shape, firmness, dry matter (%), total soluble solids, glucose, fructose, sucrose, starch, citrate, malate, vitamin C and soluble and insoluble oxalic acid. After 3 months of storage, CPPU-treated kiwifruits and the control fruit showed no difference in dry matter content, fruit firmness and total soluble solids. The results indicate that a low dosage of CPPU applied in pre-anthesis can improve fruit weight/size without any negative effect on fruit qualitative and nutritional characteristics. Copyright © 2014 Elsevier Ltd. All rights reserved.

Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B-cells

PubMed Central

Le, Anne; Lane, Andrew N.; Hamaker, Max; Bose, Sminu; Gouw, Arvin; Barbi, Joseph; Tsukamoto, Takashi; Rojas, Camilio J.; Slusher, Barbara S.; Zhang, Haixia; Zimmerman, Lisa J.; Liebler, Daniel C.; Slebos, Robbert J.C.; Lorkiewicz, Pawel K.; Higashi, Richard M.; Fan, Teresa W. M.; Dang, Chi V.

2012-01-01

Summary Because MYC plays a causal role in many human cancers, including those with hypoxic and nutrient-poor tumor microenvironments, we have determined the metabolic responses of a MYC-inducible human Burkitt lymphoma model P493 cell line to aerobic and hypoxic conditions, and to glucose deprivation, using Stable Isotope Resolved Metabolomics. Using [U-13C]-glucose as the tracer, both glucose consumption and lactate production were increased by MYC expression and hypoxia. Using [U-13C,15N]-glutamine as the tracer, glutamine import and metabolism through the TCA cycle persisted under hypoxia, and glutamine contributed significantly to citrate carbons. Under glucose deprivation, glutamine-derived fumarate, malate, and citrate were significantly increased. Their 13C labeling patterns demonstrate an alternative energy-generating glutaminolysis pathway involving a glucose-independent TCA cycle. The essential role of glutamine metabolism in cell survival and proliferation under hypoxia and glucose deficiency, makes them susceptible to the glutaminase inhibitor BPTES, and hence could be targeted for cancer therapy. PMID:22225880

Oxaloacetate supplementation increases lifespan in Caenorhabditis elegans through an AMPK/FOXO-dependent pathway

PubMed Central

Williams, David S.; Cash, Alan; Hamadani, Lara; Diemer, Tanja

2010-01-01

Summary Reduced dietary intake increases lifespan in a wide variety of organisms. It also retards disease progression. We tested whether dietary supplementation of citric acid cycle metabolites could mimic this lifespan effect. We report that oxaloacetate supplementation increased lifespan in Caenorhabditis elegans. The increase was dependent on the transcription factor, FOXO/DAF-16, and the energy sensor, AMP-activated protein kinase, indicating involvement of a pathway that is also required for lifespan extension through dietary restriction. These results demonstrate that supplementation of the citric acid cycle metabolite, oxaloacetate, influences a longevity pathway, and suggest a tractable means of introducing the health-related benefits of dietary restriction. PMID:19793063

Periconceptional Folic Acid Supplementation Benefit to Development of Early Sensory-Motor Function through Increase DNA Methylation in Rat Offspring

PubMed Central

Li, Wen; Li, Zhenshu; Li, Shou; Wang, Xinyan; Wilson, John X.; Huang, Guowei

2018-01-01

Periconceptional maternal folate levels may alter DNA methylation patterns and health outcomes in offspring. We hypothesized that maternal folic acid supplementation alters fetal neural development through DNA methylation in the fetal brain. Twenty-eight rats were randomly assigned to four groups: three groups of the female rats were fed folate-normal, folate-deficient or folate-supplemented diets from seven days before mating to delivery. In another group, folic acid supplementation diet short-period group was fed a folate-normal diet, except for 10 days (begin mating) when this group was fed a folate-supplemented diet. After delivery, the diets were changed to folate-normal diet for all four groups. The cliff avoidance and forelimb grip tests were used to assess sensory motor function of rat offspring. The results indicate that maternal folic acid supplementation improved the early development of sensory-motor function in offspring. Maternal folic acid supplementation increased the methylation potential, global DNA methylation (5-mC) and DNA methyltransferase expression and activity in the brains of the offspring. In conclusion, maternal folic acid supplementation increases DNA methylation pattern in offspring brain and improves the early development of sensory-motor function. PMID:29494536

An overview of consumer attitudes and beliefs about plant food supplements.

PubMed

Egan, Bernadette; Hodgkins, Charo; Shepherd, Richard; Timotijevic, Lada; Raats, Monique

2011-12-01

The use of dietary supplements is increasing globally and this includes the use of plant food supplements (PFS). A variety of factors may be influencing this increased consumption including the increasing number of older people in society, mistrust in conventional medicine and the perception that natural is healthy. Consumer studies in this area are limited, with a focus on dietary supplements in general, and complicated by the use of certain plant food supplements as herbal medicines. Research indicates that higher use of dietary supplements has been associated with being female, being more educated, having a higher income, being white and being older, however the drivers for consumption of supplements are complex, being influenced by both demographic and health-related factors. The aim of this paper is to provide an overview of current knowledge about the users and the determinants of usage of plant food supplements. With growing consumption of these products, the need for effective risk-benefit assessment becomes ever more important and an insight into who uses these types of products and why is an important starting point for any future science-based decisions made by policy makers, PFS manufacturers and ultimately by consumers themselves.