Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 265
Filter
1.
J Biotechnol ; 351: 23-29, 2022 Jun 10.
Article in English | MEDLINE | ID: mdl-35483474

ABSTRACT

Malic acid is one of the organic acids which is used in various industries including food and pharmaceuticals. Biotechnological production of malic acid by an efficient microorganism is highly desirable as the process will be eco-friendly and cost-effective. In this study, malic acid synthesis by Zymomonas mobilis was studied by expressing Escherichia coli malic enzyme gene under Pchap, Ptac and Ppdc promoters. The mae+ recombinants were obtained by recombineering-based genomic integration of Pchap-mae, Ptac-mae and Ppdc-mae sequences. The Ppdc promoter showed the highest expression of malic enzyme and the Pchap the lowest. However, cell growth was limited in mae+ recombinant containing Ppdc promoter. The metabolic analysis showed the highest level of malic acid in Ppdc-mae recombinant (2.84 g/L), which was about eight times higher than that in the wild type strain. The study showed that these three promoters can be used to produce organic acids in Z. mobilis.


Subject(s)
Malate Dehydrogenase , Malates , Zymomonas , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Malate Dehydrogenase/biosynthesis , Malate Dehydrogenase/genetics , Malates/metabolism , Promoter Regions, Genetic/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Zymomonas/genetics , Zymomonas/metabolism
2.
Protein Expr Purif ; 174: 105665, 2020 10.
Article in English | MEDLINE | ID: mdl-32416131

ABSTRACT

Malate dehydrogenase (MDH), which is ubiquitously occurred in nature, catalyzes the interconversion of malate and oxaloacetate. Higher plants contain multiple forms of MDH that differ in coenzyme specificity, subcellular localization and physiological function. A putative Bambusa oldhamii BoMDH cDNA was screened with the specific probe from the bamboo cDNA library. Sequence alignment shows that there's a high homology between the deduced amino acid sequence of BoMDH and MDH protein in Oryza sativa glyoxysome (92%). A 57 kDa fusion protein was expressed by IPTG induction in Escherichia coli BL21 (DE3), and an obvious MDH activity was detected in the recombinant protein. The molecular mass of recombinant BoMDH was estimated to be 120 kDa, and the subunit form was 57 kDa by denatured SDS-PAGE, indicating that BoMDH presents as a homodimer. The optimum temperature and pH for BoMDH activity were 40 °C and 9.5, respectively. The Km values of BoMDH for malate and NAD+ were 5.2 mM and 0.52 mM. The kcat/Km values of BoMDH for malate and NAD+ were 163 min-1 mM-1 and 3060 min-1 mM-1.


Subject(s)
Bambusa , Cloning, Molecular , Malate Dehydrogenase , Plant Proteins , Bambusa/enzymology , Bambusa/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Malate Dehydrogenase/biosynthesis , Malate Dehydrogenase/chemistry , Malate Dehydrogenase/genetics , Malate Dehydrogenase/isolation & purification , Plant Proteins/biosynthesis , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
3.
PLoS One ; 14(4): e0215472, 2019.
Article in English | MEDLINE | ID: mdl-30990828

ABSTRACT

Early ripening apples are usually used for fresh marketing because of short storage life, although they are with high acid and low sugar contents. Understanding the malate metabolism in fleshy fruit and underpinning process during ripening is crucial for particular crop improvement where acidity is a concern for direct consumption or further processing. In this research, a traditional Chinese apple cultivar 'Hongyu', which belongs to early ripening apple cultivar, were freshly harvested at commercial maturity stage (120 Days after full bloom) and used for different storage temperature (4°C, 20°C) and UV-C treatment (following storage at 20°C after treatment). Simple sugars (glucose, sucrose, and fructose) and organic acids (malic, and oxalic) were assessed after 14 d of storage. Compared to fruits stored at 20°C, the malate content in fruits stored at 4°C significantly higher, while it was decreased significantly in UV-C treated fruits stored at 20°C after 14 d of storage. The sugar content was almost similar throughout the UV-C-treated fruits and fruits stored at different temperature. The higher ratios of total sugars to total organic acids in UV-C treated fruits after 14 d suggest that UV-C treatment has the potential to improve the taste of early ripening apple cultivars. Considering the significant difference in malate the samples at 14 d of storage were subjected for RNA-seq analysis. Transcriptome analysis revealed that the phenomena underlying this change were governed by metabolism of malate by the regulation of NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxylase kinase (PEPCK) in apple during postharvest storage. This transcriptome profiling results have specified the transcript regulation of malate metabolism and lead to possible taste improvement without affecting the other fruit quality attributes.


Subject(s)
Food Storage , Fruit/growth & development , Gene Expression Regulation, Enzymologic/radiation effects , Gene Expression Regulation, Plant/radiation effects , Malate Dehydrogenase/biosynthesis , Malates/metabolism , Malus/growth & development , Plant Proteins/biosynthesis , Ultraviolet Rays , Gene Expression Profiling
4.
Biol Pharm Bull ; 41(3): 383-393, 2018.
Article in English | MEDLINE | ID: mdl-29491215

ABSTRACT

Cigarette smoke contains over 4800 compounds, including at least 200 toxicants or endocrine disruptors. Currently, effects of cigarette smoke on thyroid hormone (TH) levels remains to be clarified. Here, we demonstrate that cigarette smoke extract (CSE) possesses thyroid hormone properties and acts synergistically as a partial agonist for thyroid hormone receptors (TRs) in the presence of TH. In transient gene expression experiments, CSE stimulated transcriptional activity with TH in a dose-dependent manner. Stimulatory effects were observed with physiological TH concentrations, although CSE did not activate TRs without TH. CSE (5%) dissolved in phosphate-buffered saline (PBS) supplemented with 1 nM TH was approximately comparable to 3.2±0.1 and 2.3±0.2 nM of TRα1 and TRß1, respectively. To illustrate probable mechanisms of the CSE agonistic activity, effects on TR mediated transcriptional functions with cofactors were investigated. With a mammalian two-hybrid assay, CSE recruited the nuclear coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC1) to the TR. Unsaturated carbonyl compounds, acrolein, crotonaldehyde, and methyl vinyl ketone, representative constituents of CSE, retained such agonistic properties and possibly contributed to stimulatory effects. The results suggest that CSE recruits a transcriptional activator and may reinforce TH binding to the TR additively, resulting in gene expression. CSE partially agonizes TH action and may disturb the function of various nuclear hormone receptor types and their cofactors to disrupt the physiological processes.


Subject(s)
Nicotiana/adverse effects , Receptors, Thyroid Hormone/drug effects , Smoke/adverse effects , Thyroid Hormones/pharmacology , Transcription, Genetic/drug effects , Carrier Proteins/drug effects , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Malate Dehydrogenase/biosynthesis , Nerve Tissue Proteins/drug effects , Nuclear Receptor Coactivator 1/genetics , Receptors, Thyroid Hormone/genetics , Smoke/analysis , Thyroid Hormone Receptors alpha/drug effects , Thyroid Hormone Receptors alpha/genetics , Thyroid Hormone Receptors beta/drug effects , Thyroid Hormone Receptors beta/genetics , Nicotiana/chemistry
5.
Cancer Sci ; 109(6): 2036-2045, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29601126

ABSTRACT

Malic enzyme 1 (ME1) is a multifunctional protein involved in glycolysis, the citric acid cycle, NADPH production, glutamine metabolism, and lipogenesis. It is overexpressed in various cancers. We examined the expression of ME1 in 119 oral squamous cell carcinomas (OSCCs) using immunohistochemistry. Malic enzyme 1 expression was moderate to strong in 57 (48%) OSCCs and correlated with pT, pN, clinical stage, and histological grade. In 37 cases with prognostic evaluation, moderate to strong ME1 expression indicated a worse prognosis than did weak ME1 expression. Malic enzyme 1 knockdown or inactivation by lanthanide inhibited cell proliferation and motility and suppressed the epithelial-mesenchymal transition in HSC3 human OSCC cells. Knockdown of ME1 also shifted energy metabolism from aerobic glycolysis and lactate fermentation to mitochondrial oxidative phosphorylation, and the redox status from reductive to oxidative. In a mouse tumor model, lanthanide suppressed tumor growth and increased survival time. These findings reveal that ME1 is a valid target for molecular therapy in OSCC.


Subject(s)
Carcinoma, Squamous Cell/enzymology , Cytosol/enzymology , Malate Dehydrogenase/biosynthesis , Mouth Neoplasms/enzymology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Disease Progression , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Lanthanoid Series Elements/pharmacology , Malate Dehydrogenase/antagonists & inhibitors , Malate Dehydrogenase/genetics , Mouth Neoplasms/genetics , Mouth Neoplasms/pathology , Oligonucleotides, Antisense/genetics , Oxidation-Reduction/drug effects , Transplantation, Heterologous
6.
ACS Synth Biol ; 7(2): 689-695, 2018 02 16.
Article in English | MEDLINE | ID: mdl-29301074

ABSTRACT

Post-translational modifications (PTMs) play important roles in regulating a variety of biological processes. To facilitate PTM studies, the genetic code expansion strategy has been utilized to cotranslationally incorporate individual PTMs such as acetylation and phosphorylation into proteins at specific sites. However, recent studies have demonstrated that PTMs actually work together to regulate protein functions and structures. Thus, simultaneous incorporation of multiple distinct PTMs into one protein is highly desirable. In this study, we utilized the genetic incorporation systems of phosphoserine and acetyllysine to install both phosphorylation and acetylation into target proteins simultaneously in Escherichia coli. And we used this system to study the effect of coexisting acetylation and phosphorylation on malate dehydrogenase, demonstrating a practical application of this system in biochemical studies. Furthermore, we tested the mutual orthogonality of three widely used genetic incorporation systems, indicating the possibility of incorporating three distinct PTMs into one protein simultaneously.


Subject(s)
Escherichia coli Proteins , Escherichia coli , Malate Dehydrogenase , Protein Processing, Post-Translational , Acetylation , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Malate Dehydrogenase/biosynthesis , Malate Dehydrogenase/genetics , Phosphorylation/genetics
7.
Appl Biochem Biotechnol ; 184(4): 1308-1318, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29019075

ABSTRACT

Recently, malic acid has gained attention due to its potential application in food, pharmaceutical, and medical industries. In this study, the synthetic scaffold complex strategy was employed between the two key enzymes pyruvate kinase (PykF) and malic enzyme (SfcA); SH3 ligand was attached to PykF, and the SH3 domain was attached to the C-terminus of ScfA. Synthetic scaffold systems can organize enzymes spatially and temporally to increase the local concentration of intermediates. In a flask culture, the recombinant strain harboring scaffold complex produced a maximum concentration of 5.72 g/L malic acid from 10 g/L glucose. The malic acid production was significantly increased 2.1-fold from the initial culture period. Finally, malic acid production was elevated to 30.2 g in a 5 L bioreactor from recombinant strain XL-1 blue.


Subject(s)
Escherichia coli Proteins , Escherichia coli , Malate Dehydrogenase , Malates/metabolism , Pyruvate Kinase , Recombinant Fusion Proteins , src Homology Domains , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Malate Dehydrogenase/biosynthesis , Malate Dehydrogenase/genetics , Pyruvate Kinase/biosynthesis , Pyruvate Kinase/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics
8.
Nucleic Acids Res ; 43(22): e156, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26250114

ABSTRACT

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNA(Pyl) to create tRNA(Pyl-opt) with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (N(ϵ)-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNA(Pyl-opt) facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNA(Pyl-opt) with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNA(Pyl-opt) had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNA(Pyl-opt) should be an excellent replacement of wild-type tRNA(Pyl) for future ncAA incorporation by PylRS enzymes.


Subject(s)
Amino Acids/metabolism , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Directed Molecular Evolution , Escherichia coli/genetics , Escherichia coli/metabolism , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Histones/biosynthesis , Histones/genetics , Humans , Malate Dehydrogenase/biosynthesis , Malate Dehydrogenase/genetics , Transfer RNA Aminoacylation
9.
Radiat Oncol ; 10: 145, 2015 Jul 16.
Article in English | MEDLINE | ID: mdl-26173780

ABSTRACT

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) is an aggressive tumor that is treated with a combination of chemotherapy and radiation if the patient is not a candidate for surgery. Predictive biomarkers for response to radiotherapy are lacking in this patient population, making it a non-tailored therapy regimen with unknown outcome. Twenty to 30 % of NSCLC harbor an activating mutation in KRAS that may confer radioresistance. We hypothesized that mutant KRAS can regulate glutamine metabolism genes in NSCLC and maintain tumor redox balance through transamination reactions that generate cytosolic NADPH via malic enzyme 1 (ME1), which may contribute to radioresistance. FINDINGS: A doxycycline-inducible mouse model of KRAS (G12D) driven NSCLC and patient data was analyzed from multiple publicly accessible databases including TCGA, CCLE, NCBI GEO and Project Achilles. ME1 expression was found to be mutant KRAS associated in both a NSCLC mouse model and human NSCLC cancer cell lines. Perturbing glutamine metabolism sensitized mutant KRAS, but not wild-type KRAS NSCLC cell lines to radiation treatment. NSCLC survival analysis revealed that patients with elevated ME1 and GOT1 expression had significantly worse outcomes after radiotherapy, but this was not seen after chemotherapy alone. CONCLUSIONS: KRAS driven glutamine metabolism genes, specifically ME1 and GOT1 reactions, may be a predictive marker and potential therapeutic target for radiotherapy in NSCLC.


Subject(s)
Carcinoma, Non-Small-Cell Lung/radiotherapy , Genes, ras , Lung Neoplasms/radiotherapy , Malate Dehydrogenase/biosynthesis , Neoplasm Proteins/biosynthesis , Animals , Aspartate Aminotransferase, Cytoplasmic/biosynthesis , Aspartate Aminotransferase, Cytoplasmic/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Databases, Factual , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Glutamine/metabolism , Humans , Kaplan-Meier Estimate , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Malate Dehydrogenase/analysis , Malate Dehydrogenase/genetics , Mice , Mice, Transgenic , Mutation, Missense , NADP/metabolism , Neoplasm Proteins/analysis , Neoplasm Proteins/genetics , Oxidation-Reduction , Point Mutation , Prognosis , RNA Interference , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , RNA, Small Interfering/genetics , Radiation Tolerance/genetics , Recombinant Fusion Proteins/biosynthesis , Tumor Stem Cell Assay , Up-Regulation
10.
FEMS Microbiol Lett ; 362(9)2015 May.
Article in English | MEDLINE | ID: mdl-25862576

ABSTRACT

As C4-dicarboxylic acids could replace C4-petrochemicals, the reductive tricarboxylic acid (TCA) pathway was overexpressed in Pichia pastoris for production of the C4-dicarboxylic acids. Three expression cassettes which carried the pyruvate carboxylase gene (pc), the cytoplasmic malate dehydrogenase gene (mdh1) and the retargeted fumarase gene (Tfum) were integrated into the chromosomal DNA of P. pastoris GS115 alone or jointly. Multicopy integrations were screened using quantitative PCR for C4-dicarboxylic acid overaccumulation. The results showed that the highest titer in 96 h of fumaric, malic and succinic acid (0.76, 42.28 and 9.42 g l(-1)) was obtained by co-expression of pc and mdh1 in P. pastoris. This is the first report about multiple genes engineered in P. pastoris for C4-dicarboxylic acid production. The strain Pp-PC-MDH1, moreover, has a significant potential to produce malic acid in aerobic conditions.


Subject(s)
Citric Acid Cycle , Dicarboxylic Acids/metabolism , Fumarate Hydratase/biosynthesis , Malate Dehydrogenase/biosynthesis , Metabolic Engineering/methods , Pyruvate Carboxylase/biosynthesis , Citric Acid Cycle/genetics , Fumarate Hydratase/genetics , Fumarates/metabolism , Genome, Bacterial , Homologous Recombination , Malate Dehydrogenase/genetics , Malates/metabolism , Methanol/metabolism , Pichia/genetics , Pichia/physiology , Pyruvate Carboxylase/genetics
11.
Tumour Biol ; 36(8): 6211-21, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25753478

ABSTRACT

Malic enzyme 1 (ME1) links the glycolytic and citric acid cycles and is important for NADPH production, glutamine metabolism, and lipogenesis. Recently, its deregulation has been implicated in the progression of various cancers. However, the role of ME1 in the progression of hepatocellular carcinoma (HCC) remains unclear. In this study, we utilized short hairpin RNA-mediated gene silencing to investigate the biological effects of ME1 depletion in HCC and determined its prognostic significance in HCC. ME1 expression was examined by real-time (RT)-PCR and Western blot using five HCC cell lines and one normal liver cell line. We used polyethylenimine nanoparticles to deliver a short hairpin RNA to induce cessation of ME1 expression in HCC cells. Changes in NADPH production and reactive oxygen species (ROS) production were studied. Metastatic potentials of HCC cells were evaluated in vitro. Furthermore, we evaluated the protein level of ME1 in para-tumor and cancerous tissues of 65 HCC patients with detailed clinical, pathological, and clinical follow-up data. Patients' survivals were further assessed as well. Upregulated ME1 expression was observed in HCC cell lines. Downregulation of ME1 attenuated NADPH production and stimulated ROS production. Silencing ME1 was noted to inhibit migratory and invasive properties of HCC cells by inducing the E-cadherin expression and decreasing of N-cadherin and vimentin expression in a ROS-dependent pathway. Overexpression of ME1 was observed in a major fraction of HCC samples. Higher level of ME1 in tumors was significantly associated with reduced overall survival (Kaplan-Meier analysis, P = 0.024) and reduced progression-free survival (Kaplan-Meier analysis, P = 0.011). Inhibition of ME1 expression decreases HCC metastasis via suppression of epithelial-mesenchymal transition (EMT) processes in ROS-induced pathways. ME1 overexpression associates with unfavorable prognoses in patients with HCC, suggesting that ME1 is a poor prognostic predictor of hepatocellular carcinoma.


Subject(s)
Biomarkers, Tumor/biosynthesis , Carcinoma, Hepatocellular/genetics , Epithelial-Mesenchymal Transition/genetics , Liver Neoplasms/genetics , Malate Dehydrogenase/biosynthesis , Aged , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Liver Neoplasms/pathology , Malate Dehydrogenase/genetics , Male , Middle Aged , Prognosis , RNA, Small Interfering , Reactive Oxygen Species/metabolism
12.
Metab Eng ; 27: 1-9, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25447640

ABSTRACT

To obtain fast growing oil-rich microalgal strains has been urgently demanded for microalgal biofuel. Malic enzyme (ME), which is involved in pyruvate metabolism and carbon fixation, was first characterized in microalgae here. Overexpression of Phaeodactylum tricornutum ME (PtME) significantly enhanced the expression of PtME and its enzymatic activity in transgenic P. tricornutum. The total lipid content in transgenic cells markedly increased by 2.5-fold and reached a record 57.8% of dry cell weight with a similar growth rate to wild type, thus keeping a high biomass. The neutral lipid content was further increased by 31% under nitrogen-deprivation treatment, still 66% higher than that of wild type. Transgenic microalgae cells exhibited obvious morphological changes, as the cells were shorter and thicker and contained larger oil bodies. Immuno-electron microscopy targeted PtME to the mitochondrion. This study markedly increased the oil content in microalgae, suggesting a new route for developing ideal microalgal strains for industrial biodiesel production.


Subject(s)
Algal Proteins , Diatoms , Gene Expression , Genetic Engineering/methods , Lipid Metabolism , Malate Dehydrogenase , Algal Proteins/biosynthesis , Algal Proteins/genetics , Diatoms/enzymology , Diatoms/genetics , Malate Dehydrogenase/biosynthesis , Malate Dehydrogenase/genetics , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism
13.
Genet Mol Res ; 14(4): 17673-83, 2015 Dec 22.
Article in English | MEDLINE | ID: mdl-26782412

ABSTRACT

In this experiment, the test materials were 'Red Fuji' apple trees grafted onto three interstocks (No. 53, No. 111, and No. 236), which were chosen from SH40 seeding interstocks. The content of malic acid, the enzyme activities, and the expression of genes related to malic acid metabolism were determined during fruit development.The results showed that malic acid content in the ripe fruit on interstock No. 53 was higher than that in the interstock No. 111 fruit. The malate dehydrogenase (NAD-MDH) activity in apples on interstock No. 53 was highest on Day 30, Day 100, and Day 160 after bloom, and the malic enzyme (NADP-ME) activity in apples on interstock No. 111 was higher than in the interstock No. 53 fruit from Day 70 to Day 100 after bloom. The relative expression of NAD-MDH genes in interstock No. 53 fruit was higher than in No. 236 fruit on Day 100 after bloom, but the relative expression of NADP-ME in No. 236 interstock fruit was lower than in No. 53 fruit. The relative expression of NAD-MDH genes in No. 53 interstock fruit was highest on Day 160 after bloom. This might have been the main reason for the difference in the accumulation of malic acid in the ripe apples.There was a positive correlation between the relative expression of phosphoenolpyruvate carboxylase (PEPC) and the malic acid content of the fruit, and the content of malic acid in the apples was affected by the PEPC activity during the early developmental stage.


Subject(s)
Fruit/genetics , Malate Dehydrogenase/biosynthesis , Malates/metabolism , Malus/genetics , Fruit/enzymology , Fruit/growth & development , Gene Expression Regulation, Plant , Malate Dehydrogenase/genetics , Malus/enzymology , Malus/growth & development
14.
Biochem Biophys Res Commun ; 456(2): 676-82, 2015 Jan 09.
Article in English | MEDLINE | ID: mdl-25511696

ABSTRACT

Metabolic modifications during the developmental period can extend longevity. We found that malic enzyme (Men) overexpression during the larval period lengthened the lifespan of Drosophila. Men overexpression by S106-GeneSwitch-Gal4 driver increased pyruvate content and NADPH/NADP(+) ratio but reduced triglyceride, glycogen, and ATP levels in the larvae. ROS levels increased unexpectedly in Men-overexpressing larvae. Interestingly, adults exposed to larval Men-overexpression maintained ROS tolerance with enhanced expression levels of glutathione-S-transferase D2 and thioredoxin-2. Our results suggest that metabolic changes mediated by Men during development might be related to the control of ROS tolerance and the longevity of Drosophila.


Subject(s)
Drosophila Proteins/biosynthesis , Drosophila melanogaster/growth & development , Longevity/physiology , Malate Dehydrogenase/biosynthesis , Adenosine Triphosphate/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Glutathione Transferase/metabolism , Glycogen/metabolism , Larva/enzymology , Larva/genetics , Larva/growth & development , Longevity/genetics , Malate Dehydrogenase/genetics , NADP/metabolism , Pyruvic Acid/metabolism , Reactive Oxygen Species/metabolism , Thioredoxins/metabolism , Triglycerides/metabolism
15.
Cancer Lett ; 356(2 Pt A): 217-23, 2015 Jan 28.
Article in English | MEDLINE | ID: mdl-24614286

ABSTRACT

The tricarboxylic acid (TCA) cycle is a central route for oxidative metabolism. Besides being responsible for the production of NADH and FADH2, which fuel the mitochondrial electron transport chain to generate ATP, the TCA cycle is also a robust source of metabolic intermediates required for anabolic reactions. This is particularly important for highly proliferating cells, like tumour cells, which require a continuous supply of precursors for the synthesis of lipids, proteins and nucleic acids. A number of mutations among the TCA cycle enzymes have been discovered and their association with some tumour types has been established. In this review we summarise the current knowledge regarding alterations of the TCA cycle in tumours, with particular attention to the three germline mutations of the enzymes succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase, which are involved in the pathogenesis of tumours, and to the aberrant regulation of TCA cycle components that are under the control of oncogenes and tumour suppressors.


Subject(s)
Citric Acid Cycle/genetics , Energy Metabolism/genetics , Mitochondria/pathology , Neoplasms/pathology , Aconitate Hydratase/biosynthesis , Cell Proliferation/physiology , Citric Acid Cycle/physiology , Fumarate Hydratase/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Isocitrate Dehydrogenase/genetics , Malate Dehydrogenase/biosynthesis , Mitochondria/genetics , Mitochondria/metabolism , Mutation , Neoplasms/genetics , Neoplasms/metabolism , Oxidative Phosphorylation , Prolyl Hydroxylases/metabolism , Protein Serine-Threonine Kinases/biosynthesis , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Signal Transduction/genetics , Succinate Dehydrogenase/genetics
16.
PLoS Genet ; 10(10): e1004685, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25340584

ABSTRACT

Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated.


Subject(s)
Codon, Terminator/genetics , L-Lactate Dehydrogenase/genetics , Malate Dehydrogenase/genetics , Protein Biosynthesis , Receptors, Cytoplasmic and Nuclear/genetics , Ribosomes/genetics , Fungi/genetics , HeLa Cells , Humans , Isoenzymes/biosynthesis , Isoenzymes/genetics , L-Lactate Dehydrogenase/biosynthesis , Malate Dehydrogenase/biosynthesis , Nucleotide Motifs/genetics , Peroxisome-Targeting Signal 1 Receptor , Peroxisomes/genetics , RNA, Messenger/genetics , Ustilago/genetics
17.
Mol Biochem Parasitol ; 194(1-2): 64-8, 2014.
Article in English | MEDLINE | ID: mdl-24798031

ABSTRACT

The liver fluke, Opisthorchis felineus of the Opisthorchiidae family, is a well-known causative agent of opisthorchiasis in Russia and Europe. The aim of this work was to identify genes encoding thyroid hormone receptors in O. felineus, and to analyze the expression of possible target genes in response to treatment with exogenous thyroid hormones. We identified two genes encoding thyroid hormone receptors in the O. felineus genome, THRA and THRB. The genes were differentially expressed through the life cycle. The maximal level of mRNA expression of THRA1 and THRB was observed in adult worms. Treatment of the worms with triiodothyronine and thyroxine resulted in an increase in glucose 6-phosphatase mRNA expression and a decrease in malate dehydrogenase mRNA expression, potential gene targets of thyroid hormones. These data indicate that thyroid hormone receptors may perform essential roles in physiological processes in adult O. felineus.


Subject(s)
Opisthorchis/metabolism , Receptors, Thyroid Hormone/metabolism , Animals , Gene Expression Profiling , Gene Expression Regulation/drug effects , Glucose-6-Phosphatase/biosynthesis , Malate Dehydrogenase/biosynthesis , Opisthorchis/drug effects , Opisthorchis/genetics , Receptors, Thyroid Hormone/genetics , Thyroxine/metabolism , Triiodothyronine/metabolism
18.
J Oleo Sci ; 62(5): 283-92, 2013.
Article in English | MEDLINE | ID: mdl-23648402

ABSTRACT

Hop (Humulus lupulus L.) pomace contains procyanidin-rich polyphenols, which are large oligomeric compounds of catechin. We studied the effect of high dose (1%) of dietary hop pomace polyphenols (HPs) in Otsuka Long-EvansTokushima Fatty (OLETF) rats, an animal model of type 2 diabetes. By 70 days, the rats fed HPs tended to have a lower body weight and reduced mesenteric white adipose tissue weight than the rats fed a control diet. Triglyceride levels in both plasma and liver tended to be lower in the HPs-fed group than in the control group. Dietary HPs substantially suppressed the activities of hepatic fatty acid synthetase, glucose-6-phosphate dehydrogenase, and malic enzyme, through the suppression of SREBP1c mRNA expression in OLETF rats. Moreover, in the HPs-fed group, monocyte chemotactic protein-1 (MCP-1) expression and fasting blood glucose levels at 40 days, and hemoglobin A1c (HbA1c) levels at 70 days were significantly lower than those in the control group. Thus, dietary HPs may exert an ameliorative function on hepatic fatty acid metabolism, glucose metabolism, and inflammatory response accompanying the increase of the adipose tissue mass in OLETF rats.


Subject(s)
Adipose Tissue/metabolism , Catechin/pharmacology , Diabetes Mellitus, Type 2/blood , Dietary Supplements , Fasting/blood , Humulus/chemistry , Adipose Tissue/pathology , Animals , Blood Glucose/metabolism , Catechin/chemistry , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/pathology , Disease Models, Animal , Glucosephosphate Dehydrogenase/biosynthesis , Malate Dehydrogenase/biosynthesis , Male , Rats , Sterol Regulatory Element Binding Protein 1/biosynthesis , Triglycerides/blood
19.
Metab Eng ; 19: 10-6, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23707987

ABSTRACT

The yeast Torulopsis glabrata CCTCC M202019, which is used for industrial pyruvate production, was chosen to explore the suitability of engineering this multi-vitamin auxotrophic yeast for increased malate production. Various metabolic engineering strategies were used to manipulate carbon flux from pyruvate to malate: (i) overexpression of pyruvate carboxylase and malate dehydrogenase; (ii) identification of the bottleneck in malate production by model iNX804; (iii) simultaneous overexpression of genes RoPYC, RoMDH and SpMAE1. Using these strategies, 8.5gL(-1) malate was accumulated in the engineered strain T.G-PMS, which was about 10-fold greater than that of the control strain T.G-26. The results presented here suggest that T. glabrata CCTCC M202019 is a promising candidate for industrial malate production.


Subject(s)
Candida glabrata/metabolism , Malates/metabolism , Metabolic Engineering , Candida glabrata/genetics , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Malate Dehydrogenase/biosynthesis , Malate Dehydrogenase/genetics , Pyruvate Carboxylase/biosynthesis , Pyruvate Carboxylase/genetics , Pyruvic Acid/metabolism
20.
J Neurochem ; 118(3): 440-8, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21623795

ABSTRACT

These experiments reveal for the first time that microRNAs (miRNAs) mediate oxidant regulated expression of a mitochondrial tricarboxylic acid cycle gene (mdh2). mdh2 encoded malate dehydrogenase (MDH) is elevated by an unknown mechanism in brains of patients that died with Alzheimer's disease. Oxidative stress, an early and pervasive event in Alzheimer's disease, increased MDH activity and mRNA level of mdh2 by 19% and 22%, respectively, in a mouse hippocampal cell line (HT22). Post-transcriptional events underlie the change in mRNA because actinomycin D did not block the elevated mdh2 mRNA. Since miRNAs regulate gene expression post-transcriptionally, the expression of miR-743a, a miRNA predicted to target mdh2, was determined and showed a 52% reduction after oxidant treatment. Direct interaction of miR-743a with mdh2 was demonstrated with a luciferase based assay. Over-expression or inhibition of miR-743a led to a respective reduction or increase in endogenous mRNA and MDH activity. The results demonstrate that miR-743a negatively regulates mdh2 at post-transcriptional level by directly targeting the mdh2 3'UTR. The findings are consistent with the suggestion that oxidative stress can elevate the activity of MDH through miR-743a, and provide new insights into possible roles of miRNA in oxidative stress and neurodegeneration.


Subject(s)
Malate Dehydrogenase/biosynthesis , MicroRNAs/pharmacology , Mitochondria/enzymology , Oxidative Stress/physiology , 3' Untranslated Regions/genetics , Animals , Brain Chemistry/physiology , Cell Line , Enzyme Activation/drug effects , Gene Targeting , Hippocampus/drug effects , Hippocampus/enzymology , Hydrogen Peroxide/metabolism , Luciferases/metabolism , Malate Dehydrogenase/metabolism , Mice , Mice, 129 Strain , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/genetics , Transfection , Up-Regulation
SELECTION OF CITATIONS
SEARCH DETAIL
...