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Sample records for aluminum-inducible malate release

  1. Characterization of AtALMT1 expression in aluminum inducible malate release and its role for rhizotoxic stress in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Malate transporters play a critical role in aluminum (Al) tolerance responses for some plant species, such as Arabidopsis (Arabidopsis thaliana). Here we further characterize AtALMT1, an Arabidopsis malate transporter, to clarify its specific role in malate release and Al stress responses. Malate ex...

  2. Long non-coding RNA MALAT1 promotes tumour growth and metastasis in colorectal cancer through binding to SFPQ and releasing oncogene PTBP2 from SFPQ/PTBP2 complex

    PubMed Central

    Ji, Q; Zhang, L; Liu, X; Zhou, L; Wang, W; Han, Z; Sui, H; Tang, Y; Wang, Y; Liu, N; Ren, J; Hou, F; Li, Q

    2014-01-01

    Background: Metastasis associated with lung adenocarcinoma transcript-1 (MALAT1) is a functional long non-coding RNA (lncRNA), which is highly expressed in several tumours, including colorectal cancer (CRC). Its biological function and mechanism in the prognosis of human CRC is still largely under investigation. Methods: This study aimed to investigate the new effect mechanism of MALAT1 on the proliferation and migration of CRC cells in vitro and in vivo, and detect the expression of MALAT1, SFPQ (also known as PSF (PTB-associated splicing factor)), and PTBP2 (also known as PTB (polypyrimidine-tract-binding protein)) in CRC tumour tissues, followed by correlated analysis with clinicopathological parameters. Results: We found that overexpression of MALAT1 could promote cell proliferation and migration in vitro, and promote tumour growth and metastasis in nude mice. The underlying mechanism was associated with tumour suppressor gene SFPQ and proto-oncogene PTBP2. In CRC, MALAT1 could bind to SFPQ, thus releasing PTBP2 from the SFPQ/PTBP2 complex. In turn, the increased SFPQ-detached PTBP2 promoted cell proliferation and migration. SFPQ critically mediated the regulatory effects of MALAT1. Moreover, in CRC tissues, MALAT1 and PTBP2 were overexpressed, both of which were associated closely with the invasion and metastasis of CRC. However, the SFPQ showed unchanged expression either in CRC tissues or adjacent normal tissues. Conclusions: Our findings implied that MALAT1 might be a potential predictor for tumour metastasis and prognosis. Furthermore, the interaction between MALAT1 and SFPQ could be a novel therapeutic target for CRC. PMID:25025966

  3. Quercetin attenuates neuronal death against aluminum-induced neurodegeneration in the rat hippocampus.

    PubMed

    Sharma, D R; Wani, W Y; Sunkaria, A; Kandimalla, R J; Sharma, R K; Verma, D; Bal, A; Gill, K D

    2016-06-01

    Aluminum is a light weight and toxic metal present ubiquitously on earth, which has gained considerable attention due to its neurotoxic effects. It also has been linked ecologically and epidemiologically to several neurological disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), Guamanian-Parkinsonian complex and Amyotrophic lateral sclerosis (ALS). The mechanism of aluminum neurotoxicity is poorly understood, but it is well documented that aluminum generates reactive oxygen species (ROS). Enhanced ROS production leads to disruption of cellular antioxidant defense systems and release of cytochrome c (cyt-c) from mitochondria to cytosol resulting in apoptotic cell death. Quercetin (a natural flavonoid) protects it from oxidative damage and has been shown to decrease mitochondrial damage in various animal models of oxidative stress. We hypothesized that if oxidative damage to mitochondria does play a significant role in aluminum-induced neurodegeneration, and then quercetin should ameliorate neuronal apoptosis. Administration of quercetin (10mg/kg body wt/day) reduced aluminum (10mg/kg body wt/day)-induced oxidative stress (decreased ROS production, increased mitochondrial superoxide dismutase (MnSOD) activity). In addition, quercetin also prevents aluminum-induced translocation of cyt-c, and up-regulates Bcl-2, down-regulates Bax, p53, caspase-3 activation and reduces DNA fragmentation. Quercetin also obstructs aluminum-induced neurodegenerative changes in aluminum-treated rats as seen by Hematoxylin and Eosin (H&E) staining. Further electron microscopic studies revealed that quercetin attenuates aluminum-induced mitochondrial swelling, loss of cristae and chromatin condensation. These results indicate that treatment with quercetin may represent a therapeutic strategy to attenuate the neuronal death against aluminum-induced neurodegeneration. PMID:26944603

  4. Aluminum Induces Rigor within the Actin Network of Soybean Cells.

    PubMed Central

    Grabski, S.; Schindler, M.

    1995-01-01

    Aluminum is toxic to both plants and animals. Root growth and pollen-tube extension are inhibited after aluminum stress in acidic environments. Incubation of cultured neurons with aluminum results in the formation of neurofibrillar tangles reminiscent of the neural pathology observed in Alzheimer's disease. The present communication demonstrates that aluminum induces a rapid and dramatic increase in the rigidity of the actin network in soybean (Glycine max) root cells. This rigidity can be prevented by either co-incubation with sodium fluoride or magnesium, or pretreatment with cytochalasin D. It is proposed that the growth-inhibitory activity and cytotoxicity of aluminum in plants may be a consequence of a global rigor that is induced within the actin network. This rigor may result from the formation of nonhydrolyzable [Al3+-ADP] or [Al3+-ATP] complexes whose binding to actin/myosin can modify contraction. Additionally, Al3+-mediated interference with the normal kinetics of F-actin filament assembly/disassembly could precipitate subsequent disorganization of associated cytoskeletal structures and promote altered expression of cytoskeletal proteins. PMID:12228515

  5. Aluminum-induced pulmonary fibrosis: do fibers play a role?

    PubMed

    Gilks, B; Churg, A

    1987-07-01

    A 50-yr-old man with a history of 19 yr of work in the aluminum smelting industry, including 14 years in the potrooms, was found to have diffuse interstitial fibrosis, slightly more severe in the upper zones. He died of respiratory insufficiency 5 yr after initial presentation. Analysis of lung by electron optical techniques revealed 15,000,000,000 nonfibrous particles and 1,300,000,000 fibrous particles of aluminum oxide/g dry lung, values representing approximately a 1,000-fold increase over background exposure. The nonfibrous particles had a geometric mean diameter of 0.4 mu, and the fibers had a geometric mean length of 1.0 mu, a width of 0.06 mu, and an aspect ratio of 16. X-ray diffraction demonstrated alpha but not gamma aluminum oxide. These studies indicate that previous suggestions relating aluminum-induced fibrosis to the presence of gamma aluminum oxide are not correct. Although pulmonary fibrosis in this case may be a response to a very high total aluminum particle burden, the presence of large numbers of fibers raises the possibility that fibers play a role in aluminum fibrosis. PMID:3605831

  6. The metabolism of malate by cultured rat brain astrocytes

    SciTech Connect

    McKenna, M.C.; Tildon, J.T.; Couto, R.; Stevenson, J.H.; Caprio, F.J. )

    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 biphasic 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.

  7. Thermostable malate synthase of Streptomyces thermovulgaris.

    PubMed

    Goh, L L; Koh, R; Loke, P; Sim, T S

    2003-10-01

    The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40-60 degrees C) than those of scMS (28-37 degrees C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9-91.4% amino acid identities, with stMS possessing slightly more charged residues (approximately 31%) than its mesophilic counterparts (approximately 28-29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics. PMID:13680388

  8. 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. PMID:27039280

  9. Protective Effects of L-Malate against Myocardial Ischemia/Reperfusion Injury in Rats

    PubMed Central

    Ding, Shiao; Yang, Yang; Mei, Ju

    2016-01-01

    Objective. To investigate the protective effects of L-malate against myocardial ischemia/reperfusion (I/R) injury in rats. Methods. Male Sprague-Dawley rats were randomly assigned to the following groups: sham (sham), an ischemia/reperfusion (I/R) model group (model), an DMF pretreated group (DMF), and 5 L-malate pretreated groups (15, 60, 120, 240, or 480 mg/kg, gavage) before inducing myocardial ischemia. Plasma LDH, cTn-I, TNF-α, hs-CRP, SOD, and GSH-PX were measured 3 h later I/R. Areas of myocardial infarction were measured; hemodynamic parameters during I/R were recorded. Hearts were harvested and Western blot was used to quantify Nrf2, Keap1, HO-1, and NQO-1 expression in the myocardium. Results. L-malate significantly reduced LDH and cTn-I release, reduced myocardial infarct size, inhibited expression of inflammatory cytokines, and partially preserved heart function, as well as increasing antioxidant activity after myocardial I/R injury. Western blot confirmed that L-malate reduced Kelch-like ECH-associated protein 1 in ischemic myocardial tissue, upregulated expression of Nrf2 and Nrf2 nuclear translocation, and increased expression of heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1, which are major targets of Nrf2. Conclusions. L-malate may protect against myocardial I/R injury in rats and this may be associated with activation of the Nrf2/Keap1 antioxidant pathway. PMID:26941825

  10. EFFECTS OF ALUMINUM-INDUCED AGGREGATION ON THE FLUORESCENCE OF HUMIC SUBSTANCES. (R822251)

    EPA Science Inventory

    Aluminum-induced aggregates of terrestrial and aquatic humic acid standards from the International Humic Substances Society are shown to be fluorescent by means of a multiwavelength fluorescence anisotropy experiment in which the data was treated with a model for nonspherical ...

  11. Arsenic mobilization by citrate and malate from a red mud-treated contaminated soil.

    PubMed

    Castaldi, Paola; Silvetti, Margherita; Mele, Elena; Garau, Giovanni; Deiana, Salvatore

    2013-01-01

    The mobility and bioavailability of As in the soil-plant system can be affected by a number of organic acids that originate from the activity of plants and microorganisms. In this study we evaluated the ability of citrate and malate anions to mobilize As in a polluted subacidic soil (UP soil) treated with red mud (RM soil). Both anions promoted the mobilization of As from UP and RM soils, with citrate being more effective than malate. The RM treatment induced a greater mobility of As. The amounts of As released in RM and UP soils treated with 3.0 mmol L citric acid solution were 2.78 and 1.83 μmol g respectively, whereas an amount equal to 1.73 and 1.06 μmol g was found after the treatment with a 3.0 mmol L malic acid solution. The release of As in both soils increased with increasing concentration of organic acids, and the co-release of Al and Fe in solution also increased. The sequential extraction showed that Fe/Al (oxi)hydroxides in RM were the main phases involved in As binding in RM soil. Two possible mechanisms could be responsible for As solubilization: (i) competition of the organic anions for As adsorption sites and (ii) partial dissolution of the adsorbents (e.g., dissolution of iron and aluminum oxi-hydroxides) induced by citrate or malate and formation of complexes between dissolved Fe and Al and organic anions. This is the first report on the effect of malate and citrate on the As mobility in a polluted soil treated with RM. PMID:23673944

  12. 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.

  13. Aggregation states of mitochondrial malate dehydrogenase.

    PubMed Central

    Sánchez, S. A.; Hazlett, T. L.; Brunet, J. E.; Jameson, D. M.

    1998-01-01

    The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 +/- 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below 10(-9) M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium. PMID:9792106

  14. Malate transport and vacuolar ion channels in CAM plants.

    PubMed

    Cheffings, C M; Pantoja, O; Ashcroft, F M; Smith, J A

    1997-03-01

    Malate is a ubiquitous vacuolar anion in terrestrial plants that plays an important role in carbon metabolism and ionic homeostasis. In plants showing crassulacean acid metabolism (CAM), malate is accumulated as a central intermediary in the process of photosynthetic carbon assimilation, and it is also one of the major charge-balancing anions present in the vacuole. During the CAM cycle, malic acid produced as a result of dark CO(2) fixation accumulates in the vacuole at night (2 H(+) per malate), and is remobilized from the vacuole in the following light period. CAM plants thus provide a good model for studying both the mechanism and control of malate transport across the tonoplast. Thermodynamic considerations suggest that malate(2-) (the anionic species transported out of the cytosol) is passively distributed across the tonoplast. Malic acid accumulation could thus be explained by malate(2-) transport into the vacuole occurring electrophoretically in response to the transmembrane electrical potential difference established by the tonoplast H(+)-ATPase and/or H(+)-PPase. Recent studies using the patch-clamp technique have provided evidence for the existence of a vacuolar malate-selective anion channel (VMAL) in both CAM species and C(3) species. The VMAL current has a number of distinctive properties that include strong rectification (opening only at cytosolicside negative membrane potentials that would favour malate uptake into the vacuole), lack of Ca(2+) dependence, and slow activation kinetics. The kinetics of VMAL activation can be resolved into three components, consisting of an instantaneous current and two slower components with voltage-independent time constants of 0.76 s and 5.3 s in Kalanchoë daigremontiana. These characteristics suggest that the VMAL channel represents the major pathway for malate transport into the vacuole, although the strong rectification of the channel means there may be a separate, still-to-be-identified, transport mechanism for

  15. Aluminum-induced pneumoconiosis confirmed by analytical scanning electron microscopy: A case report and review of the literature.

    PubMed

    Carney, John; McAdams, Page; McCluskey, James; Roggli, Victor L

    2016-01-01

    Aluminum-induced lung injury is an uncommon, yet recognized pneumoconiosis capable of causing severe interstitial fibrosis. Important attention to the clinical history including occupational exposure is an essential component to making the correct diagnosis, despite which careful examination of the lung specimen is necessary to exclude other more common disease entities. We present a case of aluminum-induced pneumoconiosis in the setting of a bilateral lung transplant patient. Additionally, we review the literature on aluminum-induced pneumoconiosis and demonstrate the use of ancillary techniques including backscattered electron imaging and energy-dispersive spectrometry to aid in diagnosis. PMID:26895029

  16. 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

  17. Malate Synthesis and Secretion Mediated by a Manganese-Enhanced Malate Dehydrogenase Confers Superior Manganese Tolerance in Stylosanthes guianensis1

    PubMed Central

    Chen, Zhijian; Sun, Lili; Liu, Pandao; Liu, Guodao; Tian, Jiang; Liao, Hong

    2015-01-01

    Manganese (Mn) toxicity is a major constraint limiting plant growth on acidic soils. Superior Mn tolerance in Stylosanthes spp. has been well documented, but its molecular mechanisms remain largely unknown. In this study, superior Mn tolerance in Stylosanthes guianensis was confirmed, as reflected by a high Mn toxicity threshold. Furthermore, genetic variation of Mn tolerance was evaluated using two S. guianensis genotypes, which revealed that the Fine-stem genotype had higher Mn tolerance than the TPRC2001-1 genotype, as exhibited through less reduction in dry weight under excess Mn, and accompanied by lower internal Mn concentrations. Interestingly, Mn-stimulated increases in malate concentrations and exudation rates were observed only in the Fine-stem genotype. Proteomic analysis of Fine-stem roots revealed that S. guianensis Malate Dehydrogenase1 (SgMDH1) accumulated in response to Mn toxicity. Western-blot and quantitative PCR analyses showed that Mn toxicity resulted in increased SgMDH1 accumulation only in Fine-stem roots, but not in TPRC2001-1. The function of SgMDH1-mediated malate synthesis was verified through in vitro biochemical analysis of SgMDH1 activities against oxaloacetate, as well as in vivo increased malate concentrations in yeast (Saccharomyces cerevisiae), soybean (Glycine max) hairy roots, and Arabidopsis (Arabidopsis thaliana) with SgMDH1 overexpression. Furthermore, SgMDH1 overexpression conferred Mn tolerance in Arabidopsis, which was accompanied by increased malate exudation and reduced plant Mn concentrations, suggesting that secreted malate could alleviate Mn toxicity in plants. Taken together, we conclude that the superior Mn tolerance of S. guianensis is achieved by coordination of internal and external Mn detoxification through malate synthesis and exudation, which is regulated by SgMDH1 at both transcription and protein levels. PMID:25378694

  18. [Piezoresistivity of ultra-thin poly-silicon layer by aluminum-induced layer exchange].

    PubMed

    Wang, Cheng-long; Ma, Jun; Fan, Duo-wang; Xing, Da; Liu, Song-hao

    2015-02-01

    Poly-Si film, due to its favorable piezoresistive properties, has been widely used in piezoresistive sensors. The previous researches have shown that the ultra-thin poly-Si film have better piezoresistive properties than common poly-silicon film, and have promising future of application. A promising method to obtain large grained high quality poly-silicon films by low-temperature crystallization of an amorphous precursor material is the aluminum-induced layer exchange (ALILE). In this paper, ultra-thin poly-Si films were prepared by aluminum induced layer exchange (ALILE). Experimental results of Raman spectroscopy show that a narrow and symmetrical Raman peak at the wave number of about 518 cm(-1) was observed for all samples, indicating that the films were fully crystallized. XRD results show that the crystallites of ultra-thin poly-silicon layer were preferably (111) and (220) oriented. Hall affect measurements show that hole concentration of the films (p-type) were between 9 x 10(18) and 6 x 10(19) cm(-3). Restorative properties show that the piezoresistors exhibit gauge factors (GFs) up to 60, with temperature coefficients of GF (TCGF) between -0.17-0% degree C and temperature coefficients of resistance (TCR) between -0.2 and -0.1% degrees C. The study of the ultra-thin poly-Si films by ALILE is completed, and the study results lay a foundation for application of the film PMID:25970916

  19. Structural properties of a-Si films and their effect on aluminum induced crystallization

    SciTech Connect

    Tankut, Aydin; Ozkol, Engin; Karaman, Mehmet; Turan, Rasit; Canli, Sedat

    2015-10-15

    In this paper, we report the influence of the structural properties of amorphous silicon (a-Si) on its subsequent crystallization behavior via the aluminum induced crystallization (AIC) method. Two distinct a-Si deposition techniques, electron beam evaporation and plasma enhanced chemical vapor deposition (PECVD), are compared for their effect on the overall AIC kinetics as well as the properties of the final poly-crystalline (poly-Si) silicon film. Raman and FTIR spectroscopy results indicate that the PECVD grown a-Si films has higher intermediate-range order, which is enhanced for increased hydrogen dilution during deposition. With increasing intermediate-range order of the a-Si, the rate of AIC is diminished, leading larger poly-Si grain size.

  20. Malate Synthase Activity in Cotton and Other Ungerminated Oilseeds

    PubMed Central

    Miernyk, Jan A.; Trelease, Richard N.; Choinski, John S.

    1979-01-01

    Extracts from several species and varieties of ungerminated cotton seeds plus homogenates from 18 other oilseeds (representing 11 different families) were examined for malate synthase and isocitrate lyase activity. Malate synthase activities in the various cotton seeds ranged from 35 to 129% of the units per dry seed weight found in Deltapine 16 cotton. For other oilseeds, the range was from 0.3 to 58% of Deltapine 16 cotton. Castor bean (Ricinus communis L.) had the least activity per mg dry weight (12-fold lower than the next lowest species), while Pima cotton (Gossypium barbadense L.) had the highest level (8.53 units). On a per seed basis, these values were 15 and 747 nanomoles per minute. Malate synthase activity was measurable in all seed types examined, whereas isocitrate lyase could not be detected in any of the seeds. We suggest that synthesis of malate synthase during seed development is universal among oilseeds in the absence of glyoxylate-cycle-associated isocitrate lyase activity. PMID:16660858

  1. Compartmentation of Malate in Relation to Ion Absorption in Beet

    PubMed Central

    Osmond, C. B.; Laties, George G.

    1969-01-01

    Malate in beet discs treated in different salt solutions was labeled by a 30 min pulse of 14CO2, and subsequent changes in specific activity were followed for several hr. In treatments which resulted in net acid synthesis in response to excess cation absorption, malate specific activity fell slowly after removal of 14CO2. In solutions where no net acid synthesis occurred, and from which cation and anion were absorbed equally, malate specific activity fell rapidly when 14CO2 was removed. The foregoing suggests that the net synthesis of organic acids in response to excess cation absorption leads to the removal of organic anions from cytoplasmic metabolic pools as counter-ions in salt transport to the vacuole. The latter hypothesis was further examined by direct measurement of the distribution of labeled malate between cytoplasm and vacuole using the wash-exchange method of compartmental analysis, previously described for inorganic ions. The method satisfied the criterion of exchange specificity necessary for this purpose. Much higher retention of label in the cytoplasm was observed in KCl solutions (no net synthesis) than in K2SO4 solutions (net synthesis) after 3 hr 14CO2 fixation and subsequent wash-exchange. The observed distribution is consistent with the rapid removal of organic anions to the vacuole during net acid synthesis. The significance of organic acid transport in relation to metabolism is discussed. PMID:16657035

  2. Malate Synthesis by Dark Carbon Dioxide Fixation in Leaves 1

    PubMed Central

    Levi, Carolyn; Perchorowicz, John T.; Gibbs, Martin

    1978-01-01

    The rates of dark CO2 fixation and the label distribution in malate following dark 14CO2 fixation in a C-4 plant (maize), a C-3 plant (sunflower), and two Crassulacean acid metabolism plants (Bryophyllum calycinum and Kalanchoë diagremontianum leaves and plantlets) are compared. Within the first 30 minutes of dark 14CO2 fixation, leaves of maize, B. calycinum, and sunflower, and K. diagremontianum plantlets fix CO2 at rates of 1.4, 3.4, 0.23, and 1.0 μmoles of CO2/mg of chlorophyll· hour, respectively. Net CO2 fixation stops within 3 hours in maize and sunflower, but Crassulaceans continue fixing CO2 for the duration of the 23-hour experiment. A bacterial procedure using Lactobacillus plantarum ATCC No. 8014 and one using malic enzyme to remove the β-carboxyl (C4) from malate are compared. It is reported that highly purified malic enzyme and the bacterial method provide equivalent results. Less purified malic enzyme may overestimate the label in C4 as much as 15 to 20%. The contribution of carbon atom 1 of malate is between 18 and 21% of the total carboxyl label after 1 minute of dark CO2 fixation. Isotopic labeling in the two carboxyls approached unity with time. The rate of increase is greatest in sunflower leaves and Kalanchoë plantlets. In addition, Kalanchoë leaves fix 14CO2 more rapidly than Kalanchoë plantlets and the equilibration of the malate carboxyls occurs more slowly. The rates of fixation and the randomization are tissue-specific. The rate of fixation does not correlate with the rate of randomization of isotope in the malate carboxyls. PMID:16660319

  3. Malate and malate-channel antibodies inhibit electrogenic and ATP-dependent citrate transport across the tonoplast of citrus juice cells.

    PubMed

    Ratajczak, Rafael; Lüttge, Ulrich; Gonzalez, Pedro; Etxeberria, Ed

    2003-11-01

    Citrus juice cells accumulate high levels of citric acid in their vacuoles when compared to other organic ions including malate. Uptake of citrate into tonoplast vesicles from Citrus juice cells was investigated in the presence of malate, and after incubation with antibodies raised against the vacuolar malate-specific channel of Kalanchoë diagremontiana leaves. Antibodies against the vacuolar malate channel immunoreacted with a protein of similar size in tonoplast extracts from three Citrus varieties differing in citric acid content. Malate channel antibodies inhibited both delta MicroH(+)-dependent and delta MicroH(+)-independent ATP-dependent citrate transport, indicating common domains in both transport systems and to the malate-specific channel of Kalanchoë diagremontiana leaves. Malate strongly inhibited electrogenic citrate transport, whereas ATP-dependent citrate uptake was less affected. Kinetic analysis of citrate transport in the presence of malate confirmed the existence of two citrate transport mechanisms and indicated that both citrate and malate share a common transport channel across the tonoplast of Citrus juice cells. PMID:14658383

  4. 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.

  5. Prognostic value of a newly identified MALAT1 alternatively spliced transcript in breast cancer

    PubMed Central

    Meseure, Didier; Vacher, Sophie; Lallemand, François; Alsibai, Kinan Drak; Hatem, Rana; Chemlali, Walid; Nicolas, Andre; De Koning, Leanne; Pasmant, Eric; Callens, Celine; Lidereau, Rosette; Morillon, Antonin; Bieche, Ivan

    2016-01-01

    Background: Epigenetic deregulation is considered as a new hallmark of cancer. The long non-coding RNA MALAT1 has been implicated in several cancers; however, its role in breast cancer is still little known. Methods: We used RT–PCR, in situ hybridisation, and RPPA methods to quantify (i) the full-length (FL) and an alternatively spliced variant (Δsv) of MALAT1, and (ii) a panel of transcripts and proteins involved in MALAT1 pathways, in a large series of breast tumours from patients with known clinical/pathological status and long-term outcome. Results: MALAT1 was overexpressed in 14% (63/446) of the breast tumours. MALAT1-overexpressed tumour epithelial cells showed marked diffuse nuclear signals and numerous huge nuclear speckles. Screening of the dbEST database led to the identification of Δsv-MALAT1, a major alternatively spliced MALAT1 transcript, with a very different expression pattern compared with FL-MALAT1. This alternative Δsv-MALAT1 transcript was mainly underexpressed (18.8%) in our breast tumour series. Multivariate analysis showed that alternative Δsv-MALAT1 transcript is an independent prognostic factor. Δsv-MALAT1 expression was associated with alterations of the pre-mRNAs alternative splicing machinery, and of the Drosha-DGCR8 complex required for non-coding RNA biogenesis. Alternative Δsv-MALAT1 transcript expression was associated to YAP protein status and with an activation of the PI3K-AKT pathway. Conclusions: Our results reveal a complex expression pattern of various MALAT1 transcript variants in breast tumours, and suggest that this pattern of expressions should be taken into account to evaluate MALAT1 as predictive biomarker and therapeutic target. PMID:27172249

  6. 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

  7. Long noncoding RNA MALAT1 as a potential therapeutic target in osteosarcoma.

    PubMed

    Cai, Xianyi; Liu, Yunlu; Yang, Wen; Xia, Yun; Yang, Cao; Yang, Shuhua; Liu, Xianzhe

    2016-06-01

    Recent studies have revealed that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of several solid tumors. However, the function of MALAT1 in the tumorigenesis of osteosarcoma remains unknown. In the present study, levels of MALAT1 in human osteosarcoma cell lines and tissues were detected by quantitative real-time polymerase chain reaction (RT-PCR). The roles of MALAT1 in osteosarcoma were investigated by using in vitro and in vivo assays. We observed that MALAT1 expression was up-regulated in human osteosarcoma cell lines and tissues. In vitro knockdown of MALAT1 by siRNA significantly inhibited cell proliferation and migration, and induced cell cycle arrest and apoptosis in osteosarcoma cells. In addition, MALAT1 knockdown markedly suppressed the formation of tubular network structures and caused breakage of stress fibers in osteosarcoma cell lines U2OS and MNNG/HOS. Furthermore, MALAT1 knockdown delayed tumor growth in an osteosarcoma xenograft model. Specifically, we found that administration of MALAT1 siRNA decreased the protein levels of RhoA and its downstream effectors Rho-associated coiled-coil containing protein kinases (ROCKs). Taken together, these findings suggest that MALAT1 plays an oncogenic role in osteosarcoma and may be a promising therapeutic target for the treatment of osteosarcoma patients. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:932-941, 2016. PMID:26575981

  8. The BnALMT1 and BnALMT2 Genes from Rape Encode Aluminum-Activated Malate Transporters That Enhance the Aluminum Resistance of Plant Cells1

    PubMed Central

    Ligaba, Ayalew; Katsuhara, Maki; Ryan, Peter R.; Shibasaka, Mineo; Matsumoto, Hideaki

    2006-01-01

    The release of organic anions from roots can protect plants from aluminum (Al) toxicity and help them overcome phosphorus (P) deficiency. Our previous findings showed that Al treatment induced malate and citrate efflux from rape (Brassica napus) roots, and that P deficiency did not induce the efflux. Since this response is similar to the malate efflux from wheat (Triticum aestivum) that is controlled by the TaALMT1 gene, we investigated whether homologs of TaALMT1 are present in rape and whether they are involved in the release of organic anions. We isolated two TaALMT1 homologs from rape designated BnALMT1 and BnALMT2 (B. napus Al-activated malate transporter). The expression of these genes was induced in roots, but not shoots, by Al treatment but P deficiency had no effect. Several other cations (lanthanum, ytterbium, and erbium) also increased BnALMT1 and BnALMT2 expression in the roots. The function of the BnALMT1 and BnALMT2 proteins was investigated by heterologous expression in cultured tobacco (Nicotiana tabacum) cells and in Xenopus laevis oocytes. Both transfection systems showed an enhanced capacity for malate efflux but not citrate efflux, when exposed to Al. Smaller malate fluxes were also activated by ytterbium and erbium treatment. Transgenic tobacco cells grew significantly better than control cells following an 18 h treatment with Al, indicating that the expression of BnALMT1 and BnALMT2 increased the resistance of these plant cells to Al stress. This report demonstrates that homologs of the TaALMT1 gene from wheat perform similar functions in other species. PMID:17028155

  9. Metabolic Fate of the Carboxyl Groups of Malate and Pyruvate and their Influence on δ13C of Leaf-Respired CO2 during Light Enhanced Dark Respiration

    PubMed Central

    Lehmann, Marco M.; Wegener, Frederik; Barthel, Matti; Maurino, Veronica G.; Siegwolf, Rolf T. W.; Buchmann, Nina; Werner, Christiane; Werner, Roland A.

    2016-01-01

    The enhanced CO2 release of illuminated leaves transferred into darkness, termed “light enhanced dark respiration (LEDR)”, is often associated with an increase in the carbon isotope ratio of the respired CO2 (δ13CLEDR). The latter has been hypothesized to result from different respiratory substrates and decarboxylation reactions in various metabolic pathways, which are poorly understood so far. To provide a better insight into the underlying metabolic processes of δ13CLEDR, we fed position-specific 13C-labeled malate and pyruvate via the xylem stream to leaves of species with high and low δ13CLEDR values (Halimium halimifolium and Oxalis triangularis, respectively). During respective label application, we determined label-derived leaf 13CO2 respiration using laser spectroscopy and the 13C allocation to metabolic fractions during light–dark transitions. Our results clearly show that both carboxyl groups (C-1 and C-4 position) of malate similarly influence respiration and metabolic fractions in both species, indicating possible isotope randomization of the carboxyl groups of malate by the fumarase reaction. While C-2 position of pyruvate was only weakly respired, the species-specific difference in natural δ13CLEDR patterns were best reflected by the 13CO2 respiration patterns of the C-1 position of pyruvate. Furthermore, 13C label from malate and pyruvate were mainly allocated to amino and organic acid fractions in both species and only little to sugar and lipid fractions. In summary, our results suggest that respiration of both carboxyl groups of malate (via fumarase) by tricarboxylic acid cycle reactions or by NAD-malic enzyme influences δ13CLEDR. The latter supplies the pyruvate dehydrogenase reaction, which in turn determines natural δ13CLEDR pattern by releasing the C-1 position of pyruvate. PMID:27375626

  10. Metabolic Fate of the Carboxyl Groups of Malate and Pyruvate and their Influence on δ(13)C of Leaf-Respired CO2 during Light Enhanced Dark Respiration.

    PubMed

    Lehmann, Marco M; Wegener, Frederik; Barthel, Matti; Maurino, Veronica G; Siegwolf, Rolf T W; Buchmann, Nina; Werner, Christiane; Werner, Roland A

    2016-01-01

    The enhanced CO2 release of illuminated leaves transferred into darkness, termed "light enhanced dark respiration (LEDR)", is often associated with an increase in the carbon isotope ratio of the respired CO2 (δ(13)CLEDR). The latter has been hypothesized to result from different respiratory substrates and decarboxylation reactions in various metabolic pathways, which are poorly understood so far. To provide a better insight into the underlying metabolic processes of δ(13)CLEDR, we fed position-specific (13)C-labeled malate and pyruvate via the xylem stream to leaves of species with high and low δ(13)CLEDR values (Halimium halimifolium and Oxalis triangularis, respectively). During respective label application, we determined label-derived leaf (13)CO2 respiration using laser spectroscopy and the (13)C allocation to metabolic fractions during light-dark transitions. Our results clearly show that both carboxyl groups (C-1 and C-4 position) of malate similarly influence respiration and metabolic fractions in both species, indicating possible isotope randomization of the carboxyl groups of malate by the fumarase reaction. While C-2 position of pyruvate was only weakly respired, the species-specific difference in natural δ(13)CLEDR patterns were best reflected by the (13)CO2 respiration patterns of the C-1 position of pyruvate. Furthermore, (13)C label from malate and pyruvate were mainly allocated to amino and organic acid fractions in both species and only little to sugar and lipid fractions. In summary, our results suggest that respiration of both carboxyl groups of malate (via fumarase) by tricarboxylic acid cycle reactions or by NAD-malic enzyme influences δ(13)CLEDR. The latter supplies the pyruvate dehydrogenase reaction, which in turn determines natural δ(13)CLEDR pattern by releasing the C-1 position of pyruvate. PMID:27375626

  11. Almotriptan, an antimigraine agent, and its malate salt.

    PubMed

    Ravikumar, K; Sridhar, B; Krishnan, Harihara; Singh, A N

    2008-01-01

    The crystal structures of almotriptan {systematic name: N,N-dimethyl-2-[5-(pyrrolidin-1-ylsulfonylmethyl)-1H-indol-3-yl]ethanamine}, C17H25N3O2S, and almotriptan malate {systematic name: N,N-dimethyl-2-[5-(pyrrolidin-1-ylsulfonylmethyl)-1H-indol-3-yl]ethanaminium malate, C17H26N3O2S+ x C4H5O5(-), a novel selective serotonin 1B/D agonist, have been determined in order to gain further insight into the structure-activity relationships of triptans. The two structures differ in the orientation of their sulfonylpyrrolidine side chains. A comparison with other triptans reveals that molecules of almotriptan, sumatriptan, zolmitriptan and rizatriptan can adopt two principal conformations. N-H...N, N-H...O and O-H...O hydrogen bonds are responsible for the molecular packing. PMID:18216450

  12. Effects of aluminum-induced aggregation on the fluorescence of humic substances

    SciTech Connect

    Sharpless, C.M.; McGown, L.B.

    1999-09-15

    Aluminum-induced aggregates of terrestrial and aquatic humic acid standards from the International Humic Substances Society are shown to be fluorescent by means of a multiwavelength fluorescence anisotropy experiment in which the data were treated with a model for nonspherical particles. While aggregates of aquatic humic acids appear in the fluorescence signal at both short and long excitation wavelengths, aggregates of terrestrial humic acids are detected only at the long Wavelength. Furthermore, the results indicate that emission obtained at longer excitation wavelengths is representative of smaller particles. At pH 4, the aquatic humic acids appear to exist in an extended conformation, whereas the terrestrial humic acids show less extension. The size and shape of the fluorescent particles display a complex dependence on Al concentration. Both enhancement and quenching of fluorescence are observed in the total luminescence spectra upon Al addition. However, quenching is shown to be the result of decreased humic acid concentration due to precipitation by Al rather than photophysical processes.

  13. Optimal dose of zinc supplementation for preventing aluminum-induced neurotoxicity in rats.

    PubMed

    Lu, Hao; Hu, Jianyang; Li, Jing; Pang, Wei; Hu, Yandan; Yang, Hongpeng; Li, Wenjie; Huang, Chengyu; Zhang, Mingman; Jiang, Yugang

    2013-10-15

    Zinc supplementation can help maintain learning and memory function in rodents. In this study, we hypothesized that zinc supplementation could antagonize the neurotoxicity induced by aluminum in rats. Animals were fed a diet containing different doses of zinc (50, 100, 200 mg/kg) for 9 weeks, and orally administered aluminum chloride (300 mg/kg daily) from the third week for 7 consecutive weeks. Open-field behavioral test results showed that the number of rearings in the group given the 100 mg/kg zinc supplement was significantly increased compared with the group given the 50 mg/kg zinc supplement. Malondialdehyde content in the cerebrum was significantly decreased, while dopamine and 5-hydroxytryptamine levels were increased in the groups given the diet supplemented with 100 and 200 mg/kg zinc, compared with the group given the diet supplemented with 50 mg/kg zinc. The acetylcholinesterase activity in the cerebrum was significantly decreased in the group given the 100 mg/kg zinc supplement. Hematoxylin-eosin staining revealed evident pathological damage in the hippocampus of rats in the group given the diet supplemented with 50 mg/kg zinc, but the damage was attenuated in the groups given the diet supplemented with 100 and 200 mg/kg zinc. Our findings suggest that zinc is a potential neuroprotective agent against aluminum-induced neurotoxicity in rats, and the optimal dosages are 100 and 200 mg/kg. PMID:25206586

  14. Naringin protects memory impairment and mitochondrial oxidative damage against aluminum-induced neurotoxicity in rats.

    PubMed

    Prakash, Atish; Shur, Bhargabi; Kumar, Anil

    2013-09-01

    Aluminum has been indicated in neurodegenerative disorders and naringin, a bioflavonoid has been used to reduce neurotoxic effects of aluminum against aluminum chloride-induced rats. Therefore, present study has been designed to explore the possible role of naringin against aluminum-induced cognitive dysfunction and oxidative damage in rats. Aluminum (100 mg/kg) and naringin (40 and 80 mg/kg) drug treatment were administered orally for six weeks to male wistar rats. Various behavioral performance tasks, biochemical, mitochondrial oxidative parameters, and aluminum concentration in the brain were assessed. Aluminum chloride treatment significantly caused cognitive dysfunction and mitochondria oxidative damage as compared to vehicle treated control group. Besides, aluminum chloride treatment significantly increased acetyl cholinesterase activity and aluminum concentration in the brain as compared to sham. Chronic administration of naringin significantly improved cognitive performance and attenuated mitochondria oxidative damage, acetyl cholinesterase activity, and aluminum concentration in aluminum-treated rats as compared to control rats. Results of the study demonstrate neuroprotective potential of naringin against aluminum chloride-induced cognitive dysfunction and mitochondrial oxidative damage. PMID:23510099

  15. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    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 native 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

  16. 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

  17. A MALAT1/HIF-2α feedback loop contributes to arsenite carcinogenesis

    PubMed Central

    Xu, Yuan; Liu, Yi; Liu, Xinlu; Lu, Lu; Li, Jun; Wang, Qingling; Wei, Shaofeng; Shi, Le; Lu, Xiaolin; Liu, Qizhan; Zhang, Aihua

    2016-01-01

    Arsenic is well established as a human carcinogen, but the molecular mechanisms leading to arsenic-induced carcinogenesis are complex and elusive. It is also not known if lncRNAs are involved in arsenic-induced liver carcinogenesis. We have found that MALAT1, a non-coding RNA, is over-expressed in the sera of people exposed to arsenite and in hepatocellular carcinomas (HCCs), and MALAT1 has a close relation with the clinicopathological characteristics of HCC. In addition, hypoxia-inducible factor (HIF)-2α is up-regulated in HCCs, and MALAT1 and HIF-2α have a positive correlation in HCC tissues. During the malignant transformation of human hepatic epithelial (L-02) cells induced by a low concentration (2.0 μM) of arsenite, MALAT1 and HIF-2α are increased. In addition, arsenite-induced MALAT1 causes disassociation of the von Hippel-Lindau (VHL) protein from HIF-2α, therefore, alleviating VHL-mediated HIF-2α ubiquitination, which causes HIF-2α accumulation. In turn, HIF-2α transcriptionally regulates MALAT1, thus forming a positive feedback loop to ensure expression of arsenite-induced MALAT1 and HIF-2α, which are involved in malignant transformation. Moreover, MALAT1 and HIF-2α promote the invasive and metastatic capacities of arsenite-induced transformed L-02 cells and in HCC-LM3 cells. The capacities of MALAT1 and HIF-2α to promote tumor growth are validated in mouse xenograft models. In mice, arsenite induces an inflammatory response, and MALAT1 and HIF-2α are over-expressed. Together, these findings suggest that the MALAT1/HIF-2α feedback loop is involved in regulation of arsenite-induced malignant transformation. Our results not only confirm a novel mechanism involving reciprocal regulation between MALAT1 and HIF-2α, but also expand the understanding of the carcinogenic potential of arsenite. PMID:26735578

  18. Malic Acid Production by Saccharomyces cerevisiae: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export▿ †

    PubMed Central

    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.

    2008-01-01

    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, CO2-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)−1. A previously engineered glucose-tolerant, C2-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 13C-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. PMID:18344340

  19. A MALAT1/HIF-2α feedback loop contributes to arsenite carcinogenesis.

    PubMed

    Luo, Fei; Sun, Baofei; Li, Huiqiao; Xu, Yuan; Liu, Yi; Liu, Xinlu; Lu, Lu; Li, Jun; Wang, Qingling; Wei, Shaofeng; Shi, Le; Lu, Xiaolin; Liu, Qizhan; Zhang, Aihua

    2016-02-01

    Arsenic is well established as a human carcinogen, but the molecular mechanisms leading to arsenic-induced carcinogenesis are complex and elusive. It is also not known if lncRNAs are involved in arsenic-induced liver carcinogenesis. We have found that MALAT1, a non-coding RNA, is over-expressed in the sera of people exposed to arsenite and in hepatocellular carcinomas (HCCs), and MALAT1 has a close relation with the clinicopathological characteristics of HCC. In addition, hypoxia-inducible factor (HIF)-2α is up-regulated in HCCs, and MALAT1 and HIF-2α have a positive correlation in HCC tissues. During the malignant transformation of human hepatic epithelial (L-02) cells induced by a low concentration (2.0 μM) of arsenite, MALAT1 and HIF-2α are increased. In addition, arsenite-induced MALAT1 causes disassociation of the von Hippel-Lindau (VHL) protein from HIF-2α, therefore, alleviating VHL-mediated HIF-2α ubiquitination, which causes HIF-2α accumulation. In turn, HIF-2α transcriptionally regulates MALAT1, thus forming a positive feedback loop to ensure expression of arsenite-induced MALAT1 and HIF-2α, which are involved in malignant transformation. Moreover, MALAT1 and HIF-2α promote the invasive and metastatic capacities of arsenite-induced transformed L-02 cells and in HCC-LM3 cells. The capacities of MALAT1 and HIF-2α to promote tumor growth are validated in mouse xenograft models. In mice, arsenite induces an inflammatory response, and MALAT1 and HIF-2α are over-expressed. Together, these findings suggest that the MALAT1/HIF-2α feedback loop is involved in regulation of arsenite-induced malignant transformation. Our results not only confirm a novel mechanism involving reciprocal regulation between MALAT1 and HIF-2α, but also expand the understanding of the carcinogenic potential of arsenite. PMID:26735578

  20. Modulation of miR-19 in Aluminum-Induced Neural Cell Apoptosis.

    PubMed

    Zhu, Mingming; Huang, Cong; Ma, Xiao; Wu, Rui; Zhu, Weiwei; Li, Xiaoting; Liang, Zhaofeng; Deng, Feifei; Zhu, Jianyun; Xie, Wei; Yang, Xue; Jiang, Ye; Wang, Shijia; Wu, Jieshu; Geng, Shanshan; Xie, Chunfeng; Zhong, Caiyun

    2016-01-01

    Neuronal cell death is an important feature of neurodegeneration. Aluminum is associated with neurodegenerative disorders, particularly Alzheimer's disease. However, the underlying mechanisms by which aluminum induces neuronal apoptosis remain to be elucidated. miR-19 is a key miRNA implicated in regulating cell survival process, while the role of miR-19 in Alzheimer's disease has not been investigated. In the present study, we showed that Aluminum maltolate (Al-malt), a lipophilic Al complex which is a common component of human diet with the ability to facilitate the entry of Al into the brain, induced apoptosis in human neuroblastoma SH-SY5Y cells, along with downregulation of miR-19a/miR-19b, upregulation of miR-19-targeted PTEN, and alterations of its downstream apoptosis related proteins including AKT, p53, Bax, and Bcl-2. miR-19 overexpression attenuated Al-malt-induced apoptosis as well as changes in the expression of apoptosis related proteins in SH-SY5Y cells. We further revealed that exposure of rats to Al-malt for 12 weeks at doses relevant to human exposure significantly elevated Al concentrations in serum and brain tissues. Al-malt dose-dependently induced apoptosis in rat brain, as evidenced by increased caspase activation and increased TUNEL staining. Consistent with in vitro results, Al-malt reduced miR-19 expression and altered the expression of apoptotic related proteins in rat brain. Taken together, our data suggest for the first time that miR-19 modulation is critically involved in Al-induced neural cell apoptosis. Findings from this study could provide new insight into the molecular mechanisms of Al-associated neurodegenerative pathogenesis. PMID:26836165

  1. Poly-crystalline thin-film by aluminum induced crystallization on aluminum nitride substrate

    NASA Astrophysics Data System (ADS)

    Bhopal, Muhammad Fahad; Lee, Doo Won; Lee, Soo Hong

    2016-07-01

    Thin-film polycrystalline silicon (pc-Si) on foreign (non-silicon) substrates has been researched by various research groups for the production of photovoltaic cells. High quality pc-Si deposition on foreign substrates with superior optical properties is considered to be the main hurdle in cell fabrication. Metal induced crystallization (MIC) is one of the renowned techniques used to produce this quality of material. In the current study, an aluminum induced crystallization (AIC) method was adopted to produce pc-Si thin-film on aluminum nitride (AlN) substrate by a seed layer approach. Aluminum and a-Si layer were deposited using an e-beam evaporator. Various annealing conditions were used in order to investigate the AIC grown pc-Si seed layers for process optimization. The effect of thermal annealing on grain size, defects preferentially crystallographic orientation of the grains were analyzed. Surface morphology was studied using an optical microscope. Poly-silicon film with a crystallinity fraction between 95-100% and an FWHM between 5-6 cm-1 is achievable at low temperatures and for short time intervals. A grain size of about 10 micron can be obtained at a low deposition rate on an AIN substrate. Similarly, Focused ion beam (FIB) also showed that at 425 °C sample B and at 400 °C sample A were fully crystallized. The crystalline quality of pc-Si was evaluated using µ-Raman spectroscopy as a function of annealed conditions and Grazing incidence X-ray diffraction (GIXRD) was used to determine the phase direction of the pc-Si layer. The current study implicates that a poly-silicon layer with good crystallographic orientation and crystallinity fraction is achievable on AIN substrate at low temperatures and short time frames.

  2. Preclinical Evidence for the Use of Sunitinib Malate in the Treatment of Plexiform Neurofibromas

    PubMed Central

    Ferguson, Michael J.; Rhodes, Steven D.; Jiang, Li; Li, Xiaohong; Yuan, Jin; Yang, Xianlin; Zhang, Shaobo; Vakili, Saeed T.; Territo, Paul; Hutchins, Gary; Yang, Feng-Chun; Ingram, David A.; Clapp, D. Wade; Chen, Shi

    2016-01-01

    Purpose Plexiform neurofibromas (pNF) are pathognomonic nerve and soft tissue tumors of neurofibromatosis type I (NF1), which are highly resistant to conventional chemotherapy and associated with significant morbidity/mortality. Disruption of aberrant SCF/c-Kit signaling emanating from the pNF microenvironment induced the first ever objective therapeutic responses in a recent phase 2 trial. Sunitinib malate is a potent, highly selective RTK inhibitor with activity against c-Kit, PDGFR, and VEGFR, which have also been implicated in the pathogenesis of these lesions. Here, we evaluate the efficacy of sunitinib malate in a preclinical Krox20;Nf1flox/− pNF murine model. Experimental Design Proliferation, β-hexosaminidase release (degranulation), and Erk1/2 phosphorylation were assessed in sunitinib treated Nf1+/− mast cells and fibroblasts, respectively. Krox20;Nf1flox/− mice with established pNF were treated sunitinib or PBS-vehicle control for a duration of 12 weeks. pNF metabolic activity was monitored by serial [18F]DG-PET/CT imaging. Results Sunitinib suppressed multiple in vitro gain-in-functions of Nf1+/− mast cells and fibroblasts and attenuated Erk1/2 phosphorylation. Sunitinib treated Krox20;Nf1flox/− mice exhibited significant reductions in pNF size, tumor number, and FDG uptake compared to control mice. Histopathology revealed reduced tumor cellularity and infiltrating mast cells, markedly diminished collagen deposition, and increased cellular apoptosis in sunitinib treated pNF. Conclusions Collectively, these results demonstrate the efficacy of sunitinib in reducing tumor burden in Krox20;Nf1flox/− mice. These preclinical findings demonstrate the utility of inhibiting multiple RTKs in pNF and provide insights into the design of future clinical trials. PMID:26375012

  3. Malate metabolism in Hoya carnosa mitochondria and its role in photosynthesis during CAM phase III.

    PubMed

    Hong, Hoang Thi Kim; Nose, Akihiro; Agarie, Sakae; Yoshida, Takayuki

    2008-01-01

    This study investigated the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant, Hoya carnosa, in malate metabolism during CAM phase III. The mitochondria showed high malate dehydrogenase (mMDH) and aspartate amino transferase (mAST), and a significant amount of malic enzyme (mME) activities. H. carnosa readily oxidized malate via mME and mMDH in the presence of some cofactors such as thiamine pyrophosphate (TPP), coenzyme A (CoA) or NAD(+). A high respiration rate of malate oxidation was observed at pH 7.2 with NAD(+) and glutamate (Glu). Providing AST and Glu simultaneously into the respiratory medium strongly increased the rates of malate oxidation, and this oxidation was gradually inhibited by an inhibitor of alpha-ketoglutarate (alpha-KG) carrier, pyridoxal-5'-phosphate (PLP). The mitochondria readily oxidized aspartate (Asp) or alpha-KG individually with low rates, while they oxidized Asp and alpha-KG simultaneously with high rates, and this simultaneous oxidation was also inhibited by PLP. By measuring the capacity of the mitochondrial shuttle, it was found that the OAA produced via mMDH seemed not to be transported outside the mitochondria, but mAST interconverted OAA and Glu to Asp and alpha-KG, respectively, and exported them out via a malate-aspartate (malate-Asp) shuttle. The data in this research suggest that during phase III of PCK-CAM, H. carnosa mitochondria oxidized malate via both mME and the mMDH systems depending on metabolic requirements. However, malate metabolism by the mMDH system did not operate via a malate-OAA shuttle similarly to Ananas comosus mitochondria, but it operated via a malate-Asp shuttle similarly to Kalanchoë daigremontiana mitochondria. PMID:18403382

  4. Spectroscopic, thermal and structural studies on manganous malate crystals

    SciTech Connect

    Thomas, J. Lincy, A. Mahalakshmi, V.; Saban, K. V.

    2013-01-15

    Prismatic crystals of manganous malate have been prepared by controlled ionic diffusion in hydrosilica gel. The structure was elucidated using single crystal X-ray diffraction. The crystals are orthorhombic with space group Pbca. Vibrations of the functional groups were identified by the FTIR spectrum. Thermogravimetric and differential thermal analyses (TG-DTA) were carried out to explore the thermal decomposition pattern of the material. Structural information derived from FTIR and TG-DTA studies is in conformity with the single crystal XRD data.

  5. Safety Assessment of Dialkyl Malates as Used in Cosmetics.

    PubMed

    Becker, Lillian C; Bergfeld, Wilma F; Belsito, Donald V; Hill, Ronald A; Klaassen, Curtis D; Liebler, Daniel C; Marks, James G; Shank, Ronald C; Slaga, Thomas J; Snyder, Paul W; Andersen, F Alan

    2015-01-01

    The Cosmetic Ingredient Review Expert Panel (Panel) reviewed the safety of 6 dialkyl malate compounds used in cosmetics. These ingredients function mostly as skin-conditioning agents-emollients. The Panel reviewed relevant animal and human data related to the ingredients along with a previous safety assessment of malic acid. The similar structure, properties, functions, and uses of these ingredients enabled grouping them and using the available toxicological data to assess the safety of the entire group. The Panel concluded that these dialkyl maleate compounds are safe in the present practices of use and concentration as given in this safety assessment. PMID:26227891

  6. Natural antisense RNA promotes 3′ end processing and maturation of MALAT1 lncRNA

    PubMed Central

    Zong, Xinying; Nakagawa, Shinichi; Freier, Susan M.; Fei, Jingyi; Ha, Taekjip; Prasanth, Supriya G.; Prasanth, Kannanganattu V.

    2016-01-01

    The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3′ end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3′ end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3′ end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans. Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3′ end processing. PMID:26826711

  7. OVEREXPRESSION OF A NODULE-ENHANCED MALATE DEHYDROGENASE INCREASES NITROGEN FIXATION IN ALFALFA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Malate is crucial for symbiotic dinitrogen (N2) fixation, occurring in high concentrations in N2-fixing nodules as the major carbon source for bacteroid respiration. Malate also provides carbon skeletons for the assimilation of fixed nitrogen from ammonia into amino acids and is proposed to be invol...

  8. 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. PMID:26196273

  9. Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase*

    PubMed Central

    Takahashi-Íñiguez, Tóshiko; Aburto-Rodríguez, Nelly; Vilchis-González, Ana Laura; Flores, María Elena

    2016-01-01

    Malate dehydrogenase (MDH) is an enzyme widely distributed among living organisms and is a key protein in the central oxidative pathway. It catalyzes the interconversion between malate and oxaloacetate using NAD+ or NADP+ as a cofactor. Surprisingly, this enzyme has been extensively studied in eukaryotes but there are few reports about this enzyme in prokaryotes. It is necessary to review the relevant information to gain a better understanding of the function of this enzyme. Our review of the data generated from studies in bacteria shows much diversity in their molecular properties, including weight, oligomeric states, cofactor and substrate binding affinities, as well as differences in the direction of the enzymatic reaction. Furthermore, due to the importance of its function, the transcription and activity of this enzyme are rigorously regulated. Crystal structures of MDH from different bacterial sources led to the identification of the regions involved in substrate and cofactor binding and the residues important for the dimer-dimer interface. This structural information allows one to make direct modifications to improve the enzyme catalysis by increasing its activity, cofactor binding capacity, substrate specificity, and thermostability. A comparative analysis of the phylogenetic reconstruction of MDH reveals interesting facts about its evolutionary history, dividing this superfamily of proteins into two principle clades and establishing relationships between MDHs from different cellular compartments from archaea, bacteria, and eukaryotes.

  10. L-malate oxidation by the electron transport fraction of Azotobacter vinelandii.

    PubMed

    Jurtshuk, P; Bednarz, A J; Zey, P; Denton, C H

    1969-06-01

    The membrane-bound l-malate oxidoreductase of Azotobacter vinelandii strain O was found to be a flavoprotein-dependent enzyme associated with the electron transport system (R(3)) of this organism. The particulate R(3) fraction, which possessed the l-malate oxidoreductase, carried out the cyanide-sensitive oxidation of l-malate, d-lactate, reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, succinate, cytochrome c, tetramethyl-p-phenylenediamine, and p-phenylenediamine, with molecular O(2) as the terminal electron acceptor. d-Malate was not oxidized, but l-malate was oxidized to oxalacetate. Phenazine methosulfate (PMS), vitamin K(3), K(3)Fe(CN)(6), nitro blue tetrazolium, and dichloroindophenol all served as good terminal electron acceptors for the l-malate oxidoreductase. Cytochrome c was a poor electron acceptor. Extensive studies on the l-malate oxidase and PMS and K(3) reductases revealed that all were stimulated specifically by flavine adenine dinucleotide and nonspecifically by di- or trivalent cations, i.e., Ca(++), Ba(++), Mn(++), Mg(++), Fe(+++), Ni(++), and Al(+++). All these activities were markedly sensitive to ethylenediaminetetraacetate (EDTA). The V(max) values for the l-malate oxidase, PMS, and vitamin K(3) reductases were, respectively, 3.4, 15.1, and 45.5 mumoles of substrate oxidized per min per mg of protein at 37 C. Spectral studies revealed that the Azotobacter R(3) flavoprotein and cytochromes (a(2), a(1), b(1), c(4), and c(5)) were reduced by l-malate. l-Malate oxidase activity was sensitive to various inhibitors of the electron transport system, namely, p-chloromercuriphenylsulfonic acid, chlorpromazine, 2-n-heptyl-4-hydroxyquinoline-N-oxide, antimycin A, and KCN. Minor inhibitory effects were noted with the inhibitors 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione, rotenone, and Amytal. PMID:4977982

  11. Putative role of the malate valve enzyme NADP-malate dehydrogenase in H2O2 signalling in Arabidopsis.

    PubMed

    Heyno, Eiri; Innocenti, Gilles; Lemaire, Stéphane D; Issakidis-Bourguet, Emmanuelle; Krieger-Liszkay, Anja

    2014-04-19

    In photosynthetic organisms, sudden changes in light intensity perturb the photosynthetic electron flow and lead to an increased production of reactive oxygen species. At the same time, thioredoxins can sense the redox state of the chloroplast. According to our hypothesis, thioredoxins and related thiol reactive molecules downregulate the activity of H2O2-detoxifying enzymes, and thereby allow a transient oxidative burst that triggers the expression of H2O2 responsive genes. It has been shown recently that upon light stress, catalase activity was reversibly inhibited in Chlamydomonas reinhardtii in correlation with a transient increase in the level of H2O2. Here, it is shown that Arabidopsis thaliana mutants lacking the NADP-malate dehydrogenase have lost the reversible inactivation of catalase activity and the increase in H2O2 levels when exposed to high light. The mutants were slightly affected in growth and accumulated higher levels of NADPH in the chloroplast than the wild-type. We propose that the malate valve plays an essential role in the regulation of catalase activity and the accumulation of a H2O2 signal by transmitting the redox state of the chloroplast to other cell compartments. PMID:24591715

  12. Role of Exogenous Melatonin on Cell Proliferation and Oxidant/Antioxidant System in Aluminum-Induced Renal Toxicity.

    PubMed

    Karabulut-Bulan, Omur; Bayrak, Bertan Boran; Arda-Pirincci, Pelin; Sarikaya-Unal, Guner; Us, Huseyin; Yanardag, Refiye

    2015-11-01

    Aluminum has toxic potential on humans and animals when it accumulates in various tissues. It was shown in a number of studies that aluminum causes oxidative stress by free radical formation and lipid peroxidation in tissues and thus may cause damage in target organs. Although there are numerous studies investigating aluminum toxicity, biochemical mechanisms of the damage caused by aluminum have yet to be explained. Melatonin produced by pineal gland was shown to be an effective antioxidant. Since kidneys are target organs for aluminum accumulation and toxicity, we have studied the role of melatonin against aluminum-induced renal toxicity in rats. Wistar albino rats were divided into five groups. Group I served as control, and received only physiological saline; group II served as positive control for melatonin, and received ethanol and physiological saline; group III received melatonin (10 mg/kg); group IV received aluminum sulfate (5 mg/kg) and group V received aluminum sulfate and melatonin (in the same dose), injected three times a week for 1 month. Administration of aluminum caused degenerative changes in renal tissues, such as increase in metallothionein immunoreactivity and decrease in cell proliferation. Moreover, uric acid and lipid peroxidation levels and xanthine oxidase activity increased, while glutathione, catalase, superoxide dismutase, paraoxonase 1, glucose-6-phosphate dehydrogenase, and sodium potassium ATPase activities decreased. Administration of melatonin mostly prevented these symptoms. Results showed that melatonin is a potential beneficial agent for reducing damage in aluminum-induced renal toxicity. PMID:25855374

  13. Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss

    PubMed Central

    Arun, Gayatri; Diermeier, Sarah; Akerman, Martin; Chang, Kung-Chi; Wilkinson, J. Erby; Hearn, Stephen; Kim, Youngsoo; MacLeod, A. Robert; Krainer, Adrian R.; Norton, Larry; Brogi, Edi; Egeblad, Mikala; Spector, David L.

    2016-01-01

    Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT- and Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression. PMID:26701265

  14. Long non-coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling.

    PubMed

    Yao, Jin; Wang, Xiao-Qun; Li, Yu-Jie; Shan, Kun; Yang, Hong; Wang, Yang-Ning-Zhi; Yao, Mu-Di; Liu, Chang; Li, Xiu-Miao; Shen, Yi; Liu, Jing-Yu; Cheng, Hong; Yuan, Jun; Zhang, Yang-Yang; Jiang, Qin; Yan, Biao

    2016-01-01

    The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non-coding RNAs (lncRNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up-regulated in the retinas, Müller cells, and primary retinal ganglion cells (RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Müller cell activation, and RGC survival in vivo and in vitro MALAT1-CREB binding maintains CREB phosphorylation by inhibiting PP2A-mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling. PMID:26964565

  15. Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss.

    PubMed

    Arun, Gayatri; Diermeier, Sarah; Akerman, Martin; Chang, Kung-Chi; Wilkinson, J Erby; Hearn, Stephen; Kim, Youngsoo; MacLeod, A Robert; Krainer, Adrian R; Norton, Larry; Brogi, Edi; Egeblad, Mikala; Spector, David L

    2016-01-01

    Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT- and Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression. PMID:26701265

  16. Light and Acetate Regulate a Mitochondrial Malate Dehydrogenase 1

    PubMed Central

    Struck, Friedhelm; Grölz-Krug, Sabine; Boschek, Bruce; Zetsche, Klaus

    1987-01-01

    A malate dehydrogenase was purified from the unicellular green alga Chlorogonium elongatum Dangeard. The enzyme was localized in the mitochondria by immunogold electron microscopy and was found to be present on the cristae. The concentration of the enzyme is regulated by acetate and light. In cells cultured heterotrophically with acetate as carbon source the activity and the concentration of the enzyme is 5- to 6-fold higher than in autotrophic cells. In mixotrophically cultured cells (light and acetate) the enzyme level attains only half of the value of that in heterotrophic cells. Acetate induces an increase of the enzyme concentration while light has an inhibitory effect on this process. Images Fig. 2 Fig. 3 PMID:16665643

  17. Vacuolar malate uptake is mediated by an anion-selective inward rectifier.

    PubMed

    Hafke, Jens B; Hafke, Yuliya; Smith, J Andrew C; Lüttge, Ulrich; Thiel, Gerhard

    2003-07-01

    Electrophysiological studies using the patch-clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current-voltage relations of the tonoplast were dominated by a strongly inward-rectifying anion-selective channel that was active at cytoplasmic-side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage-dependent gating of a 3-pS malate-selective ion channel, which showed a half-maximal open probability at -43 mV. Also, the time-averaged unitary currents following a step to a negative voltage corresponded to the time-dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion-selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent Km of 2.5 mm malate2- activity), a selectivity sequence of fumarate2- > malate2- > Cl- > maleate2- approximately citrate3-, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 microM niflumate reversibly blocked the activity of the 3-pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 microm-2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3-pS, inward-rectifying, anion-selective channel represents the principal pathway for malate influx into the vacuole. PMID:12834407

  18. Long noncoding RNA MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability

    PubMed Central

    Yan, Caifeng; Chen, Jinfeng; Chen, Nuoqi

    2016-01-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is implicated in liver cell proliferation. However, its role in hepatic steatosis and insulin resistance remain poorly understood. The aim of this study was to investigate the effects of MALAT1 on hepatic lipid accumulation and its potential targets. As expected, MALAT1 expression is increased in hepatocytes exposed to palmitate and livers of ob/ob mice. Knockdown of MALAT1 expression dramatically suppressed palmitate-induced lipid accumulation and the increase of nuclear SREBP-1c protein in HepG2 cells. In addition, RNA immunoprecipitation and RNA pull-down assay confirmed that MALAT1 interacted with SREBP-1c to stabilize nuclear SREBP-1c protein. Finally, injection of si-MALAT1 prevented hepatic lipid accumulation and insulin resistance in ob/ob mice. In conclusion, our observations suggest that MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability. PMID:26935028

  19. Novel insight into MALAT-1 in cancer: Therapeutic targets and clinical applications

    PubMed Central

    REN, DANYANG; LI, HUIYING; LI, RENQIU; SUN, JIANMING; GUO, PIN; HAN, HUIYUN; YANG, YUEHUANG; LI, JUN

    2016-01-01

    Long non-protein-coding RNAs (lncRNAs) are emerging as important gene expression regulators that are linked to various biological processes at the post-transcriptional and transcriptional levels. lncRNAs are known to be important in cell proliferation, cell differentiation, apoptosis and metastasis. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), a novel lncRNA, is highly conserved amongst mammals. In addition, it has been considered to act as an oncogene, depending on the tumor system. An increasing number of studies have indicated that MALAT-1 may be detected in certain types of human tumors, including lung and bladder cancer and hepatocellular carcinoma. MALAT-1 silencing may be an effective therapeutic approach against tumors. The present study reviews the current knowledge on the functional role of MALAT-1 in the control of various cancers. PMID:26998053

  20. Effect of Malate-oligosaccharide Solution on Antioxidant Capacity of Endurance Athletes.

    PubMed

    Qiang, Fu

    2015-01-01

    L-malate is an important intermediate on the process of metabolism; it plays an important role in generating mitochondria ATP both under aerobic and hypoxic condition. It is easy to be absorbed and come into mitochondrion through cell membrane and promote to produce energy in mitochondrion. The purpose of this investigation is to probe into the different influence malate ingestion on blood lactate and glucose kinetics during aerobic exercise athletes; at the same time, rats were used to study the effect of malate and oligosaccharide solution on the metabolism in muscle and liver. The supplement of malate-oligosaccharide solution may improve the level of antioxidants in vivo after exercise, and subsequently increase the total antioxidant capacity and decrease the level of lipid peroxidation. At the appropriate time sports drinks can add varying degrees of motion to extend time to fatigue enhance athletic ability, speed up the recovery process after exercise, reduce fatigue. PMID:26998183

  1. Effect of Malate-oligosaccharide Solution on Antioxidant Capacity of Endurance Athletes

    PubMed Central

    Qiang, Fu

    2015-01-01

    L-malate is an important intermediate on the process of metabolism; it plays an important role in generating mitochondria ATP both under aerobic and hypoxic condition. It is easy to be absorbed and come into mitochondrion through cell membrane and promote to produce energy in mitochondrion. The purpose of this investigation is to probe into the different influence malate ingestion on blood lactate and glucose kinetics during aerobic exercise athletes; at the same time, rats were used to study the effect of malate and oligosaccharide solution on the metabolism in muscle and liver. The supplement of malate-oligosaccharide solution may improve the level of antioxidants in vivo after exercise, and subsequently increase the total antioxidant capacity and decrease the level of lipid peroxidation. At the appropriate time sports drinks can add varying degrees of motion to extend time to fatigue enhance athletic ability, speed up the recovery process after exercise, reduce fatigue. PMID:26998183

  2. Factors affecting the translocation of oxaloacetate and L-malate into rat liver mitochondria.

    PubMed

    Haslam, J M; Griffiths, D E

    1968-10-01

    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 K(m) values were 40mum 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 K(i) 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 50mum-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

  3. 13C Nuclear Magnetic Resonance Study of Acetate Incorporation into Malate During Ca2+-Uptake by Isolated Leaf Tissues 1

    PubMed Central

    Borchert, Rolf; Everett, Grover W.

    1987-01-01

    13C Nuclear magnetic resonance spectroscopy of leaflets of Gleditsia triacanthos and Albizia julibrisin was used to determine the fate of acetate taken up during the absorption of calcium from 13C-labeled Ca-acetate solution. Small amounts of acetate accumulated temporarily in the leaf tissues, but the bulk of acetate was incorporated into malate. The initial rate of malate synthesis was very low, but increased rapidly during acetate treatment and reached its maximum after 8 hours; the enzymes involved in malate synthesis thus appear to be substrate induced. Use of acetate-2-13C yielded malate labeled in C-3, indicating that vacuolar malate accumulating during Ca-uptake might be synthesized via malate synthase from acetate and glyoxalate. However, a source of glyoxalate condensing with acetate during malate synthesis could not be identified. Glycolate produced in photorespiration is an unlikely source, because glycolate-2-13C was absorbed and metabolized by the leaf tissues into products of the glycolate pathway, but was not a major precursor in malate synthesis. Malate synthesis via the glyoxalate cycle is also unlikely, because no evidence for the recycling of a 13C-labeled 4-carbon organic acid was found. Malate synthesis in the leaflets of Gleditsia and Albizia thus appears to involve the inducible condensation of acetate with a 2-carbon compound of unidentified nature and origin. PMID:16665548

  4. Aluminum inhibits neurofilament assembly, cytoskeletal incorporation, and axonal transport. Dynamic nature of aluminum-induced perikaryal neurofilament accumulations as revealed by subunit turnover.

    PubMed

    Shea, T B; Wheeler, E; Jung, C

    1997-01-01

    The mechanism by which aluminum induces formation of perikaryal neurofilament (NF) inclusions remains unclear. Aluminum treatment inhibits: 1. The incorporation of newly synthesized NF subunits into Triton-insoluble cytoskeleton of axonal neurites; 2. Their degradation and dephosphorylation; 3. Their translocation into axonal neurites. It also fosters the accumulation of phosphorylated NFs within perikarya. In the present study, we addressed the relationship among these effects. Aluminum reduced the assembly of newly synthesized NF subunits into NFs. During examination of those subunits that did assemble in the presence of aluminum, it was revealed that aluminum also interfered with transport of newly assembled NFs into axonal neurites. Similarly, a delay in axonal transport of microinjected biotinylated NF-H was observed in aluminum-treated cells. Aluminum also inhibited the incorporation of newly synthesized and microinjected subunits into the Triton-insoluble cytoskeleton within both perikarya and neurites. Once incorporated into Triton-insoluble cytoskeletons, however, biotinylated subunits were retained within perikarya of aluminum-treated cells to a greater extent than within untreated cells. Notably, these subunits were depleted in the presence and absence of aluminum within 48 h, despite the persistence of the aluminum-induced perikaryal accumulation itself, suggesting that individual NF subunits undergo turnover even within aluminum-induced perikaryal accumulations. These findings demonstrate that aluminum interferes with multiple aspects of neurofilament dynamics and furthermore leaves open the possibility that aluminum-induced perikaryal NF whorls may not represent permanent structures, but rather may require continued recruitment of cytoskeletal constituents. PMID:9437656

  5. The Long Noncoding RNA MALAT-1 Is Highly Expressed in Ovarian Cancer and Induces Cell Growth and Migration

    PubMed Central

    Zhou, Yanqing; Xu, Xiaying; Lv, Huabing; Wen, Qirong; Li, Juan; Tan, Linyu; Li, Jianqi; Sheng, Xiujie

    2016-01-01

    Background Metastasis associated in lung adenocarcinoma transcript-1 (MALAT-1) is overexpressed during cancer progression and promotes cell migration and invasion in many solid tumors. However, its role in ovarian cancer remains poorly understood. Methods Expressions of MALAT-1 were detected in 37 normal ovarian tissues and 45 ovarian cancer tissues by reverse transcription polymerase chain reaction (RT-PCR). Cell proliferation was observed by CCK-8 assay; Flow cytometry was used to measure cell cycle and apoptosis; Cell migration was detected by transwell migration and invasion assay. In order to evaluate the function of MALAT-1, shRNA combined with DNA microarray and Functional enrichment analysis were performed to determine the transcriptional effects of MALAT-1 silencing in OVCAR3 cells. RNA and protein expression were measured by qRT-PCR and Western blotting, respectively. Results We found that upregulation of MALAT-1 mRNA in ovarian cancer tissues and enhanced MALAT-1 expression was associated with FIGO stage. Knockdown of MALAT-1 expression in OVCAR3 cells inhibited cell proliferation, migration, and invasion, leading to G0/G1 cell cycle arrest and apoptosis. Overexpressed MALAT-1 expression in SKOV3 cells promoted cell proliferation, migration and invasion. Downregulation of MALAT-1 resulted in significant change of gene expression (at least 2-fold) in 449 genes, which regulate proliferation, cell cycle, and adhesion. As a consequence of MALAT-1 knockdown, MMP13 protein expression decreased, while the expression of MMP19 and ADAMTS1 was increased. Conclusions The present study found that MALAT-1 is highly expressed in ovarian tumors. MALAT-1 promotes the growth and migration of ovarian cancer cells, suggesting that MALAT-1 may be an important contributor to ovarian cancer development. PMID:27227769

  6. Hydroxycinnamoyl-CoA:Malate Hydroxycinnamoyl Transferase is Crucial for 2-O-Caffeoyl-L-Malate Biosynthesis in Red Clover and Defines a New Pathway for Hydroxycinnamoyl-Malate Ester Biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover (Trifolium pratense L.) leaves accumulate several µmol of phaselic acid [2-O-caffeoyl-L-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forages like alfalfa (Medi...

  7. Long Noncoding RNA MALAT-1 Enhances Stem Cell-Like Phenotypes in Pancreatic Cancer Cells

    PubMed Central

    Jiao, Feng; Hu, Hai; Han, Ting; Yuan, Cuncun; Wang, Lei; Jin, Ziliang; Guo, Zhen; Wang, Liwei

    2015-01-01

    Cancer stem cells (CSCs) play a vital role in tumor initiation, progression, metastasis, chemoresistance, and recurrence. The mechanisms that maintain the stemness of these cells remain largely unknown. Our previous study indicated that MALAT-1 may serve as an oncogenic long noncoding RNA in pancreatic cancer by promoting epithelial-mesenchymal transition (EMT) and regulating CSCs markers expression. More significantly, there is emerging evidence that the EMT process may give rise to CSCs, or at least cells with stem cell-like properties. Therefore, we hypothesized that MALAT-1 might enhance stem cell-like phenotypes in pancreatic cancer cells. In this study, our data showed that MALAT-1 could increase the proportion of pancreatic CSCs, maintain self-renewing capacity, decrease the chemosensitivity to anticancer drugs, and accelerate tumor angiogenesis in vitro. In addition, subcutaneous nude mouse xenografts revealed that MALAT-1 could promote tumorigenicity of pancreatic cancer cells in vivo. The underlying mechanisms may involve in increased expression of self-renewal related factors Sox2. Collectively, we for the first time found the potential effects of MALAT-1 on the stem cell-like phenotypes in pancreatic cancer cells, suggesting a novel role of MALAT-1 in tumor stemness, which remains to be fully elucidated. PMID:25811929

  8. 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. PMID:26512004

  9. Streptococcus pyogenes Malate Degradation Pathway Links pH Regulation and Virulence

    PubMed Central

    Paluscio, Elyse

    2015-01-01

    The ability of Streptococcus pyogenes to infect different niches within its human host most likely relies on its ability to utilize alternative carbon sources. In examining this question, we discovered that all sequenced S. pyogenes strains possess the genes for the malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised of four genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding a malate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression of maeP and maeE is induced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Furthermore, both maePE and maeKR are repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malate utilization requires the PTS transporter EI enzyme (PtsI), as a PtsI– mutant fails to express the ME genes and is unable to utilize malate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP–, MaeK–, and MaeR– mutants were attenuated for virulence, whereas a MaeE– mutant showed enhanced virulence compared to that of the wild type. Taken together, these data show that ME contributes to S. pyogenes' carbon source repertory, that malate utilization is a highly regulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptake and utilization contribute to the adaptive pH response, and ME can influence the outcome of infection. PMID:25583521

  10. Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles

    PubMed Central

    Miyagawa, Ryu; Tano, Keiko; Mizuno, Rie; Nakamura, Yo; Ijiri, Kenichi; Rakwal, Randeep; Shibato, Junko; Masuo, Yoshinori; Mayeda, Akila; Hirose, Tetsuro; Akimitsu, Nobuyoshi

    2012-01-01

    MALAT-1 noncoding RNA is localized to nuclear speckles despite its mRNA-like characteristics. Here, we report the identification of several key factors that promote the localization of MALAT-1 to nuclear speckles and also provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm. RNAi-mediated repression of the nuclear speckle proteins, RNPS1, SRm160, or IBP160, which are well-known mRNA processing factors, resulted in the diffusion of MALAT-1 to the nucleoplasm. We demonstrated that MALAT-1 contains two distinct elements directing transcripts to nuclear speckles, which were also capable of binding to RNPS1 in vitro. Depletion of MALAT-1 represses the expression of several genes. Taken together, our results suggest that RNPS1, SRm160, and IBP160 contribute to the localization of MALAT-1 to nuclear speckles, where MALAT-1 could be involved in regulating gene expression. PMID:22355166

  11. Long Noncoding RNA MALAT1 Controls Cell Cycle Progression by Regulating the Expression of Oncogenic Transcription Factor B-MYB

    PubMed Central

    Tripathi, Vidisha; Shen, Zhen; Chakraborty, Arindam; Giri, Sumanprava; Freier, Susan M.; Wu, Xiaolin; Zhang, Yongqing; Gorospe, Myriam; Prasanth, Supriya G.; Lal, Ashish; Prasanth, Kannanganattu V.

    2013-01-01

    The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p53 and its target genes. The cell cycle defects observed in MALAT1-depleted cells are sensitive to p53 levels, indicating that p53 is a major downstream mediator of MALAT1 activity. Furthermore, MALAT1-depleted cells display reduced expression of B-MYB (Mybl2), an oncogenic transcription factor involved in G2/M progression, due to altered binding of splicing factors on B-MYB pre-mRNA and aberrant alternative splicing. In human cells, MALAT1 promotes cellular proliferation by modulating the expression and/or pre-mRNA processing of cell cycle–regulated transcription factors. These findings provide mechanistic insights on the role of MALAT1 in regulating cellular proliferation. PMID:23555285

  12. LncRNA MALAT1 exerts oncogenic functions in lung adenocarcinoma by targeting miR-204

    PubMed Central

    Li, Jipeng; Wang, Jianhua; Chen, Yin; Li, Shanfeng; Jin, Mingwei; Wang, Huaying; Chen, Zhe; Yu, Wanjun

    2016-01-01

    Accumulating evidence indicates that the lncRNAs play a critical role in cancer progression and metastasis. In this study, we found that MALAT1 upregulation was associated with larger tumor size and lymph-node metastasis, and also correlated with shorter overall survival of lung adenocarcinoma patients. Furthermore, MALAT1 promotes EMT and metastasis of lung adenocarcinoma cells in vitro and in vivo. In particular, MALAT1 upregulated the expression of miR-204 target gene SLUG through competitively ‘spongeing’ miR-204. In summary we unveil a branch of the MALAT1/miR-204/SLUG pathway that regulates the progression of lung adenocarcinoma. PMID:27294002

  13. Intermolecular interactions of the malate synthase of Paracoccidioides spp

    PubMed Central

    2013-01-01

    Background The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal

  14. The efficacy of topical ivermectin versus malation 0.5% lotion for the treatment of scabies.

    PubMed

    Goldust, Mohamad; Rezaee, Elham

    2013-05-01

    Objective: There are different medications for the treatment of scabies but the treatment of choice is still controversial. This study aimed at comparing the efficacy of topical ivermectin versus malation 0.5% lotion for the treatment of scabies. Methods: In total, 340 patients with scabies were enrolled, and randomized into two groups: the first group received 1% ivermectin applied topically to the affected skin and the second group received topical malation 0.5% lotion and were told to apply this twice with 1 week interval. Treatment was evaluated at intervals of 2 and 4 weeks, and if there was treatment failure at the 2-week follow-up, treatment was repeated. Results: Two application of topical ivermectin provided a cure rate of 67.6% at the 2-week follow-up, which increased to 85.2% at the 4-week follow-up after repeating the treatment. Treatment with two applications of malation 0.5% lotion was effective in 44.1% of patients at the 2-week follow-up, which increased to 67.6% at the 4-week follow-up after this treatment was repeated. Conclusion:Two application of ivermectin was as effective as single applications of malation 0.5% lotion at the 2-week follow-up. After repeating the treatment, ivermectin was superior to malation 0.5% lotion at the 4-week follow up. PMID:23472617

  15. Kinetic studies of the uptake of aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro.

    PubMed Central

    Marra, E; Passarella, S; Casamassima, E; Perlino, E; Doonan, S; Quagliariello, E

    1985-01-01

    Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system. PMID:4015628

  16. Selective permeability of rat liver mitochondria to purified malate dehydrogenase isoenzymes in vitro.

    PubMed Central

    Passarella, S; Marra, E; Doonan, S; Quagliariello, E

    1980-01-01

    1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied. PMID:7236231

  17. Protective effects of low-intensity pulsed ultrasound on aluminum-induced cerebral damage in Alzheimer's disease rat model

    NASA Astrophysics Data System (ADS)

    Lin, Wei-Ting; Chen, Ran-Chou; Lu, Wen-Wei; Liu, Shing-Hwa; Yang, Feng-Yi

    2015-04-01

    The protein expressions of neurotrophic factors can be enhanced by low-intensity pulsed ultrasound (LIPUS) stimulation in the brain. The purpose of this study was to demonstrate the protective effect of LIPUS stimulation against aluminum-induced cerebral damage in Alzheimer's disease rat model. LIPUS was administered 7 days before each aluminum chloride (AlCl3) administration, and concomitantly given with AlCl3 daily for a period of 6 weeks. Neurotrophic factors in hippocampus were measured by western blot analysis. Behavioral changes in the Morris water maze and elevated plus maze were examined in rats after administration of AlCl3. Various biochemical analyses were performed to evaluate the extent of brain damages. LIPUS is capable of prompting levels of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF) in rat brain. AlCl3 administration resulted in a significant increase in the aluminum concentration, acetylcholinesterase activity and beta-amyloid (Aβ) deposition in AlCl3 treated rats. LIPUS stimulation significantly attenuated aluminum concentration, acetylcholinesterase activity, Aβ deposition and karyopyknosis in AlCl3 treated rats. Furthermore, LIPUS significantly improved memory retention in AlCl3-induced memory impairment. These experimental results indicate that LIPUS has neuroprotective effects against AlCl3-induced cerebral damages and cognitive dysfunction.

  18. Controlled aluminum-induced crystallization of an amorphous silicon thin film by using an oxide-layer diffusion barrier

    NASA Astrophysics Data System (ADS)

    Hwang, Ji-Hyun; Kwak, Hyunmin; Kwon, Myeung Hoi

    2014-03-01

    Aluminum-induced crystallization (AIC) of amorphous silicon with an Al2O3 diffusion barrier was investigated for controlling Si crystallization and preventing layer exchange during the annealing process. An Al2O3 layer was deposited between the a-Si and the Al films (a-Si/Al2O3/Al/Glass) and was blasted with an air spray gun with alumina beads to form diffusion channels between the Si and the Al layers. During the annealing process, small grain Si x Al seeds were formed at the channels. Then, the Al2O3 diffusion barrier was restructured to close the channels and prevent further diffusion of Al atoms into the a-Si layer. A polycrystalline Si film with (111), (220) and (311) crystallization peaks in the X-ray diffraction pattern was formed by annealing at 560 °C in a conventional furnace. That film showed a p-type semiconducting behavior with good crystallinity and a large grain size of up to 14.8 µm. No layer conversion occurred between the Si and the Al layers, which had been the fundamental obstacle to the applications in the crystallization of a-Si films by using the AIC method.

  19. Protective effects of low-intensity pulsed ultrasound on aluminum-induced cerebral damage in Alzheimer's disease rat model

    PubMed Central

    Lin, Wei-Ting; Chen, Ran-Chou; Lu, Wen-Wei; Liu, Shing-Hwa; Yang, Feng-Yi

    2015-01-01

    The protein expressions of neurotrophic factors can be enhanced by low-intensity pulsed ultrasound (LIPUS) stimulation in the brain. The purpose of this study was to demonstrate the protective effect of LIPUS stimulation against aluminum-induced cerebral damage in Alzheimer's disease rat model. LIPUS was administered 7 days before each aluminum chloride (AlCl3) administration, and concomitantly given with AlCl3 daily for a period of 6 weeks. Neurotrophic factors in hippocampus were measured by western blot analysis. Behavioral changes in the Morris water maze and elevated plus maze were examined in rats after administration of AlCl3. Various biochemical analyses were performed to evaluate the extent of brain damages. LIPUS is capable of prompting levels of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF) in rat brain. AlCl3 administration resulted in a significant increase in the aluminum concentration, acetylcholinesterase activity and beta-amyloid (Aβ) deposition in AlCl3 treated rats. LIPUS stimulation significantly attenuated aluminum concentration, acetylcholinesterase activity, Aβ deposition and karyopyknosis in AlCl3 treated rats. Furthermore, LIPUS significantly improved memory retention in AlCl3-induced memory impairment. These experimental results indicate that LIPUS has neuroprotective effects against AlCl3-induced cerebral damages and cognitive dysfunction. PMID:25873429

  20. LncRNA MALAT1 enhances oncogenic activities of EZH2 in castration-resistant prostate cancer

    PubMed Central

    Wang, Liguo; Zhao, Yu; Sun, Zhifu; Karnes, R. Jeffrey; Zhang, Jun; Huang, Haojie

    2015-01-01

    The Polycomb protein enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in advanced human prostate cancer (PCa), especially in lethal castration-resistant prostate cancer (CRPC). However, the signaling pathways that regulate EZH2 functions in PCa remain incompletely defined. Using EZH2 antibody-based RNA immunoprecipitation-coupled high throughput sequencing (RIP-seq), we demonstrated that EZH2 binds to MALAT1, a long non-coding RNA (lncRNA) that is overexpressed during PCa progression. GST pull-down and RIP assays demonstrated that the 3′ end of MALAT1 interacts with the N-terminal of EZH2. Knockdown of MALAT1 impaired EZH2 recruitment to its target loci and upregulated expression of EZH2 repressed genes. Further studies indicated that MALAT1 plays a vital role in EZH2-enhanced migration and invasion in CRPC cell lines. Meta-analysis and RT-qPCR of patient specimens demonstrated a positive correlation between MALAT1 and EZH2 expression in human CRPC tissues. Finally, we showed that MALAT1 enhances expression of PRC2-independent target genes of EZH2 in CRPC cells in culture and patient-derived xenografts. Together, these data indicate that MALAT1 may be a crucial RNA cofactor of EZH2 and that the EZH2-MALAT1 association may provide a new avenue for development new strategies for treatment of CRPC. PMID:26516927

  1. Long non-coding RNA MALAT-1 overexpression predicts tumor recurrence of hepatocellular carcinoma after liver transplantation.

    PubMed

    Lai, Ming-chun; Yang, Zhe; Zhou, Lin; Zhu, Qian-qian; Xie, Hai-yang; Zhang, Feng; Wu, Li-ming; Chen, Lei-ming; Zheng, Shu-sen

    2012-09-01

    Metastasis-associated lung adenocarcinoma transcript 1(MALAT1), a long non-coding RNA (lncRNA), is up-regulated in many solid tumors and associated with cancer metastasis and recurrence. However, its role in hepatocellular carcinoma (HCC) remains poorly understood. In the present study, we evaluated the expression of MALAT1 by quantitative real-time PCR in 9 liver cancer cell lines and 112 HCC cases including 60 cases who received liver transplantation (LT) with complete follow-up data. Moreover, small interfering RNA (siRNA) was used to inhibit MALAT1 expression to investigate its biological role in tumor progression. We found that MALAT1 was up-regulated in both cell lines and clinical tissue samples. Patients with high expression level of MALAT1 had a significantly increased risk of tumor recurrence after LT, particularly in patients who exceeded the Milan criteria. On multivariate analysis, MALAT1 was an independent prognostic factor for predicting HCC recurrence (hazard ratio, 3.280, P = 0.003).In addition, inhibition of MALAT1 in HepG2 cells could effectively reduce cell viability, motility, invasiveness, and increase the sensitivity to apoptosis. Our data suggest that lncRNA MALAT1 play an important role in tumor progression and could be a novel biomarker for predicting tumor recurrence after LT and serve as a promising therapeutic target. PMID:21678027

  2. LncRNA MALAT1 enhances oncogenic activities of EZH2 in castration-resistant prostate cancer.

    PubMed

    Wang, Dejie; Ding, Liya; Wang, Liguo; Zhao, Yu; Sun, Zhifu; Karnes, R Jeffrey; Zhang, Jun; Huang, Haojie

    2015-12-01

    The Polycomb protein enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in advanced human prostate cancer (PCa), especially in lethal castration-resistant prostate cancer (CRPC). However, the signaling pathways that regulate EZH2 functions in PCa remain incompletely defined. Using EZH2 antibody-based RNA immunoprecipitation-coupled high throughput sequencing (RIP-seq), we demonstrated that EZH2 binds to MALAT1, a long non-coding RNA (lncRNA) that is overexpressed during PCa progression. GST pull-down and RIP assays demonstrated that the 3' end of MALAT1 interacts with the N-terminal of EZH2. Knockdown of MALAT1 impaired EZH2 recruitment to its target loci and upregulated expression of EZH2 repressed genes. Further studies indicated that MALAT1 plays a vital role in EZH2-enhanced migration and invasion in CRPC cell lines. Meta-analysis and RT-qPCR of patient specimens demonstrated a positive correlation between MALAT1 and EZH2 expression in human CRPC tissues. Finally, we showed that MALAT1 enhances expression of PRC2-independent target genes of EZH2 in CRPC cells in culture and patient-derived xenografts. Together, these data indicate that MALAT1 may be a crucial RNA cofactor of EZH2 and that the EZH2-MALAT1 association may provide a new avenue for development new strategies for treatment of CRPC. PMID:26516927

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. [Physicochemical, catalytic, and regulatory properties of malate dehydrogenase from Rhodovulum steppense bacteria, strain A-20s].

    PubMed

    Eprintsev, A T; Falaleeva, M I; Parfenova, I V; Liashchenko, M S; Kompantseva, E I; Tret'iakova, A Iu

    2014-01-01

    The physicochemical, regulatory, and kinetic properties of malate dehydrogenase (EC 1.1.1.37) from haloalkaliphilic purple nonsulfur Rhodovulum steppense bacteria, strain A-20s, were studied. The malate dehydrogenase (MDH) preparation with a specific activity of 0.775 ± 0.113 U/mg protein was obtained in an electrophoretically homogeneous state using multistep purification. Using homogenous preparations, the molecular weight and the Michaelis constant of the enzyme were determined; the effects of metal ions, the temperature effect, and the thermal stability of the MDH were studied. The dimer structure of the enzyme was demonstrated by DS-Na-electrophoresis. PMID:25739304

  5. Development and Validation of HPTLC Method for the Estimation of Almotriptan Malate in Tablet Dosage Form.

    PubMed

    Suneetha, A; Syamasundar, B

    2010-09-01

    A new, simple, precise and accurate high performance thin layer chromatographic method has been proposed for the determination of almotriptan malate in a tablet dosage form. The drug was separated on aluminum plates precoated with silica gel 60 GF(254) with butanol:acetic acid:water (3:1:1) was used as mobilephase. Quantitative analysis was performed by densitometric scanning at 300 nm. The method was validated for linearity, accuracy, precision and robustness. The calibration plot was linear over the range of 100-700 ng/band for almotriptan malate. The method was successfully applied to the analysis of drug in a pharmaceutical dosage form. PMID:21694997

  6. 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. PMID:26773544

  7. Reciprocal regulation of Hsa-miR-1 and long noncoding RNA MALAT1 promotes triple-negative breast cancer development.

    PubMed

    Jin, Chuan; Yan, Bingchuan; Lu, Qin; Lin, Yanmin; Ma, Lei

    2016-06-01

    Recent studies demonstrated that long noncoding RNAs (lncRNAs) have a critical role in the regulation of cancer progression and metastasis. However, little is known about the mechanism through which metastasis-associated lung adencarcinoma transcript 1 (MALAT1) exerts its oncogenic activity, and the interaction between MALAT1 and microRNA remains largely unknown. In the present study, we reported that MALAT1 was upregulated in triple-negative breast cancer (TNBC) tissues. Knockdown of MALAT1 inhibited proliferation, motility, and increased apoptosis in vitro. In vivo study indicated that knockdown of MALAT1 inhibited tumor growth and metastasis. Patients with high MALAT1 expression had poorer overall survival time than those with low MALAT1 expression. In addition, our findings demonstrate a reciprocal negative control relationship between MALAT1 and miR-1: downregulation of MALAT1 increased expression of microRNA-1 (miR-1), while overexpression of miR-1 decreased MALAT1 expression. Slug was identified as a direct target of miR-1. We proposed that MALAT1 exerted its function through the miR-1/slug axis. In summary, we proposed that MALAT1 may be a target for TNBC therapy. PMID:26676637

  8. L-malate enhances the gene expression of carried proteins and antioxidant enzymes in liver of aged rats.

    PubMed

    Zeng, X; Wu, J; Wu, Q; Zhang, J

    2015-01-01

    Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. To investigate the antioxidant mechanism of L-malate in the mitochondria, we analyzed the change in gene expression of two malate-aspartate shuttle (MAS)-related carried proteins (AGC, aspartate/glutamate carrier and OMC, oxoglutarate/malate carrier) in the inner mitochondrial membrane, and three antioxidant enzymes (CAT, SOD, and GSH-Px) in the mitochondria. The changes in gene expression of these proteins and enzymes were examined by real-time RT-PCR in the heart and liver of aged rats treated with L-malate. L-malate was orally administered in rats continuously for 30 days using a feeding atraumatic needle. We found that the gene expression of OMC and GSH-Px mRNA in the liver increased by 39 % and 38 %, respectively, in the 0.630 g/kg L-malate treatment group than that in the control group. The expression levels of SOD mRNA in the liver increased by 39 %, 56 %, and 78 % in the 0.105, 0.210, and 0.630 g/kg L-malate treatment groups, respectively. No difference were observed in the expression levels of AGC, OMC, CAT, SOD, and GSH-Px mRNAs in the heart of rats between the L-malate treatment and control groups. These results predicted that L-malate may increase the antioxidant capacity of mitochondria by enhancing the expression of mRNAs involved in the MAS and the antioxidant enzymes. PMID:25194133

  9. INFLUENCE OF ENHANCED MALATE DEHYDROGENASE EXPRESSION BY ALFALFA ON DIVERSITY OF RHIZOBACTERIA AND SOIL NUTRIENT AVAILABILITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic alfalfa over-expressing a nodule-enhanced malate dehydrogenase (neMDH) cDNA and untransformed alfalfa plants were grown at the same field site and rhizosphere soils collected after 53 weeks of plant growth. These alfalfa lines differ in the amount and composition of root organic acids pro...

  10. CLYBL is a polymorphic human enzyme with malate synthase and β-methylmalate synthase activity

    PubMed Central

    Strittmatter, Laura; Li, Yang; Nakatsuka, Nathan J.; Calvo, Sarah E.; Grabarek, Zenon; Mootha, Vamsi K.

    2014-01-01

    CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/β-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to β-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/β-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown. PMID:24334609

  11. 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. PMID:177206

  12. Homochiral Cu(II) and Ni(II) malates with tunable structural features

    NASA Astrophysics Data System (ADS)

    Zavakhina, Marina S.; Samsonenko, Denis G.; Virovets, Alexander V.; Dybtsev, Danil N.; Fedin, Vladimir P.

    2014-02-01

    Four new homochiral metal-organic frameworks (MOFs) based on S-malate anions and N-donor linkers of different length have been prepared under solvothermal conditions. [Cu(mal)(bpy)]·H2O (1), [Cu(mal)(bpe)]·2H2O (2), [Ni(mal)(bpy)]·1.3CH3OH (3) and [Ni(mal)(bpe)]·4H2O (4) (mal=S-malate, bpy=4,4‧-bipyridil, bpe=trans-1,2-bis(4-pyridyl)ethylene) were characterized by a number of analytical methods including powder X-ray diffraction, elemental, thermogravimetric analyses, IR spectroscopy. Compounds 1-3 were structurally characterized by X-ray crystallography. The absence of the chiral ligand racemization under synthetic conditions was unambiguously confirmed by polarimetry experiments. Compounds 1 and 2 contain metal-malate layered motives, connected by N-donor linkers and contribute to the family of isoreticular Cu(II) malates and tartrates [Cu(mal)L] and [Cu(tart)L], (tart=tartrate; L=ditopic rigid organic ligand). The Ni-based compounds 3 and 4 share 1D chiral {Ni(mal)} motives and possess novel type of the chiral framework, previously unknown for chiral carboxylates. The linear N-donor linkers connect these chiral chains, thus controlling the channel diameter and guest accessible volume of the homochiral structure, which exceeds 60 %.

  13. Escherichia coli d-Malate Dehydrogenase, a Generalist Enzyme Active in the Leucine Biosynthesis Pathway*

    PubMed Central

    Vorobieva, Anastassia A.; Khan, Mohammad Shahneawz; Soumillion, Patrice

    2014-01-01

    The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB− strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (l(+)-tartrate, d-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution. PMID:25160617

  14. Homochiral Cu(II) and Ni(II) malates with tunable structural features

    SciTech Connect

    Zavakhina, Marina S.; Samsonenko, Denis G.; Virovets, Alexander V.; Dybtsev, Danil N.; Fedin, Vladimir P.

    2014-02-15

    Four new homochiral metal–organic frameworks (MOFs) based on S-malate anions and N-donor linkers of different length have been prepared under solvothermal conditions. [Cu(mal)(bpy)]·H{sub 2}O (1), [Cu(mal)(bpe)]·2H{sub 2}O (2), [Ni(mal)(bpy)]·1.3CH{sub 3}OH (3) and [Ni(mal)(bpe)]·4H{sub 2}O (4) (mal=S-malate, bpy=4,4′-bipyridil, bpe=trans-1,2-bis(4-pyridyl)ethylene) were characterized by a number of analytical methods including powder X-ray diffraction, elemental, thermogravimetric analyses, IR spectroscopy. Compounds 1–3 were structurally characterized by X-ray crystallography. The absence of the chiral ligand racemization under synthetic conditions was unambiguously confirmed by polarimetry experiments. Compounds 1 and 2 contain metal-malate layered motives, connected by N-donor linkers and contribute to the family of isoreticular Cu(II) malates and tartrates [Cu(mal)L] and [Cu(tart)L], (tart=tartrate; L=ditopic rigid organic ligand). The Ni-based compounds 3 and 4 share 1D chiral (Ni(mal)) motives and possess novel type of the chiral framework, previously unknown for chiral carboxylates. The linear N-donor linkers connect these chiral chains, thus controlling the channel diameter and guest accessible volume of the homochiral structure, which exceeds 60 %. - Graphical abstract: Four new homochiral metal–organic frameworks are built from Ni{sup 2+} or Cu{sup 2+} cations, S-malate anions and N-donor linkers of different length, which controls the size of pores and guest accessible volume of the homochiral structure. Display Omitted - Highlights: • Four new homohiral metal–organic frameworks based on Ni{sup 2+} and Cu{sup 2+}. • Cu(II)–malate layers and Ni(II)–malate chains are connected by N-donor linkers. • N-donor linkers of different length control the size of pores.

  15. How fumarase recycles after the malate --> fumarate reaction. Insights into the reaction mechanism.

    PubMed

    Rose, I A

    1998-12-22

    Recycling of yeast fumarase to permit repetition of its reaction chemistry requires two proton transfers and two conformational changes, in pathways that are different in detail but thematically similar in the two directions. In the malate --> fumarate direction, simple anions such as acetate accelerate the fumarate-off step producing E(H(f)), a fumarate-specific isoform that retains the C3R-proton of malate. Fumarate specificity is shown with S-2,3-dicarboxyaziridine, which is competitive vs fumarate and noncompetitive with malate as substrate. The steady-state level of E(H(f)), based on Kii (S-2,3-dicarboxyaziridine), is increased by D2O and decreased by imidazole acting as a general acid for conversion of E(H(f)) to E(H(f))H. E(H(f))H is fumarate-specific as shown by the inhibition pattern with ClO4-. The pKa of this step is approximately 7.25 based on the pH dependence of Kii (ClO4-). A conformational change occurs next as shown by high sensitivity of k(cat) but not k(cat)/Km, to the microviscosogen, glycerol, and change to a nonspecific isoform, E(H(mf))H, probably the same species formed in the fumarate --> malate direction from malate-specific intermediates by a different conformational change. Malate enters the cycle by reaction with E(H(mf))H and returns to E(m)H x malate after a second conformational change. When fumarate-off is slow, as in low anion medium, malate itself becomes an activator of malate --> fumarate. This effect occurs with changes in inhibition patterns suggestive of the bypass of the slow E(f) --> E(mf) conversion in favor of direct formation of E(mf) when free fumarate is formed. 3-Nitro-2-hydroxypropionate, a strong inhibitor of fumarase [Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 4772-4780] in its carbanion form, is competitive with both malate and fumarate. Therefore, 3-nitro-2-hydroxypropionic acid interacts with E(H(mf))H and not with E(m) or E(f) isoforms. Occurrence of two different conformational changes in

  16. 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. PMID:23485710

  17. Plastidial NAD-dependent malate dehydrogenase is critical for embryo development and heterotrophic metabolism in Arabidopsis.

    PubMed

    Beeler, Seraina; Liu, Hung-Chi; Stadler, Martha; Schreier, Tina; Eicke, Simona; Lue, Wei-Ling; Truernit, Elisabeth; Zeeman, Samuel C; Chen, Jychian; Kötting, Oliver

    2014-03-01

    In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis. PMID:24453164

  18. 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

  19. The long noncoding RNA MALAT1 promotes tumor-driven angiogenesis by up-regulating pro-angiogenic gene expression

    PubMed Central

    Tee, Andrew E.; Liu, Bing; Song, Renhua; Li, Jinyan; Pasquier, Eddy; Cheung, Belamy B.; Jiang, Cizhong; Marshall, Glenn M.; Haber, Michelle; Norris, Murray D.; Fletcher, Jamie I.; Dinger, Marcel E.; Liu, Tao

    2016-01-01

    Neuroblastoma is the most common solid tumor during early childhood. One of the key features of neuroblastoma is extensive tumor-driven angiogenesis due to hypoxia. However, the mechanism through which neuroblastoma cells drive angiogenesis is poorly understood. Here we show that the long noncoding RNA MALAT1 was upregulated in human neuroblastoma cell lines under hypoxic conditions. Conditioned media from neuroblastoma cells transfected with small interfering RNAs (siRNA) targeting MALAT1, compared with conditioned media from neuroblastoma cells transfected with control siRNAs, induced significantly less endothelial cell migration, invasion and vasculature formation. Microarray-based differential gene expression analysis showed that one of the genes most significantly down-regulated following MALAT1 suppression in human neuroblastoma cells under hypoxic conditions was fibroblast growth factor 2 (FGF2). RT-PCR and immunoblot analyses confirmed that MALAT1 suppression reduced FGF2 expression, and Enzyme-Linked Immunosorbent Assays revealed that transfection with MALAT1 siRNAs reduced FGF2 protein secretion from neuroblastoma cells. Importantly, addition of recombinant FGF2 protein to the cell culture media reversed the effects of MALAT1 siRNA on vasculature formation. Taken together, our data suggest that up-regulation of MALAT1 expression in human neuroblastoma cells under hypoxic conditions increases FGF2 expression and promotes vasculature formation, and therefore plays an important role in tumor-driven angiogenesis. PMID:26848616

  20. Sensitivity of the plant vacuolar malate channel to pH, Ca2+ and anion-channel blockers.

    PubMed

    Pantoja, O; Smith, J A C

    2002-03-01

    The organic anion malate is accumulated in the central vacuole of most plant cells. Malate has several important roles in plant vacuoles, such as the maintenance of charge balance and pH regulation, as an osmolyte involved in the generation of cell turgor, and as a storage form of CO2. Transport of malate across the vacuolar membrane is important for the regulation of cytoplasmic pH and the control of cellular metabolism, particularly in plants showing crassulacean acid metabolism (CAM), in which large fluxes of malate occur during the day/night cycle. By applying the patch-clamp technique, in the whole-vacuole configuration, to isolated vacuoles from leaf mesophyll cells of the CAM plant Kalanchoë daigremontiana, we studied the regulation of the vacuolar malate channel by pH and Ca2+, as well as its sensitivity to anion-channel blockers. Malate currents were found to be insensitive to Ca2+ on the cytoplasmic side of the membrane over a range from approximately 10(-8) M to 10(-4) M. In contrast, decreasing cytoplasmic pH below 7.5 had a significant modulatory effect on channel activity, reducing malate currents by 40%, whereas increasing cytoplasmic pH above 7.5 resulted in no change in current. Several known Cl?-channel blockers inhibited the vacuolar malate currents: niflumic acid and indanoyloxyacetic acid (IAA-94) proved to be the most effective inhibitors, exerting half-maximal effects at concentrations of approximately 20 mM, suggesting that the plant vacuolar malate channel may share certain similarities with other classes of known anion channels. PMID:11891587

  1. Inhibition of the long non-coding RNA MALAT1 suppresses tumorigenicity and induces apoptosis in the human ovarian cancer SKOV3 cell line

    PubMed Central

    LIU, SHIPING; JIANG, XUAN; LI, WEIHUA; CAO, DONGYAN; SHEN, KENG; YANG, JIAXIN

    2016-01-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a 8,000 nucleotide-long, spliced non-coding RNA, which has been reported to be deregulated in several tumors. However, to the best of our knowledge, the role of MALAT1 in ovarian cancer has not been previously investigated. The aim of the present study was to investigate the effect of MALAT1 inhibition on the tumorigenity of SKOV3 cells. First, stable MALAT1-knockdown ovarian cancer cells and control cells were established using lentivirus-mediated artificial micro RNA interference in order to investigate the effect of MALAT1 inhibition on cell viability, clonability, migration, invasion and apoptosis in vitro. In addition, the effect of MALAT1 on cell growth in nude mice was assessed. To identify the possible targets of MALAT1, total RNA was extracted from MALAT1-knockdown cells and control cells and a microarray analysis was performed. The results showed that MALAT1 inhibition significantly suppressed tumorigenity in vitro and in vivo (P<0.01). Compared with the control cells, 921 genes in the MALAT1-knockdown cells were deregulated by at least two-fold. The results of the reverse transcription-quantitative polymerase chain reaction showed that 19 of the 20 genes selected for validation confirmed the deregulation indicated by the microarray analysis. The findings define a major oncogenic role for MALAT1, which may offer an attractive novel target for therapeutic intervention in ovarian cancer. PMID:27313681

  2. Disequilibrium in the malate dehydrogenase reaction in rat liver mitochondria in vivo

    PubMed Central

    Heath, D. F.; Phillips, J. C.

    1972-01-01

    1. When [2-14C]pyruvate is injected into rats the C3-position of liver glutamate becomes more heavily labelled than the C2-position, thus establishing that oxaloacetate and fumarate are not in equilibrium in rat liver mitochondria in vivo. The amount of disequilibrium was shown to be simply related to the value that the C3-label/C2-label ratio would have were no label recycled. This ratio, z, was calculated for post-absorptive rats in environmental temperatures of 20° and 30°C from determinations of the distribution of label within glutamate 1, 3 and 10min after intravenous injection of [2-14C]pyruvate. The values of z (best estimate and range) were 1.65 (1.60–1.69) in rats at 20°C and 2.43 (2.23–2.63) in rats at 30°C. These values of z imply the following rates of interconversion in mitochondria of fumarate and oxaloacetate (in terms of the oxaloacetate→citrate flux, R) in rats at 20°C: [Formula: see text] and in rats at 30°C: [Formula: see text] 2. The kinetic parameters of malate dehydrogenase and fumarate hydratase and the intramitochondrial concentrations of NAD+ and NADH under (as far as could be judged) conditions in vivo were collated. From them and the best estimates of R now available were calculated the rates of interconversion of fumarate, malate and oxaloacetate required to give the found values of z. These rates showed that the fumarate hydratase reaction was nearly in equilibrium, but that the malate dehydrogenase reaction was considerably out of equilibrium. The calculations also led to the following conclusions. 3. In livers of rats at 20° and 30°C mitochondrial malate concentrations were respectively about 5 and 1.5 times mean cellular concentrations. 4. Mitochondrial oxaloacetate concentrations were less than 0.2 of the mean cellular concentrations. They were also only 0.65 and 0.55 of the equilibrium concentrations for the malate dehydrogenase reaction in rats at 20° and 30°C respectively. 5. Malate dehydrogenase activity was low

  3. High expression of lncRNA MALAT1 suggests a biomarker of poor prognosis in colorectal cancer

    PubMed Central

    Zheng, Hong-Tu; Shi, De-Bing; Wang, Yu-Wei; Li, Xin-Xiang; Xu, Ye; Tripathi, Pratik; Gu, Wei-Lie; Cai, Guo-Xiang; Cai, San-Jun

    2014-01-01

    Objective: This study sought to investigate the role of the long noncoding RNA MALAT1 in the prognosis of stage II/III colorectal cancer (CRC) patients. Methods: The expression of MALAT1 was evaluated in cancer tissues from 146 stage II/III CRC patients undergoing radical resection and 23 paired normal colonic mucosa samples using quantitative real-time reverse transcriptase PCR. Differences in the expression of MALAT1 between 23 CRC and paired normal colonic mucosa samples were analysed with the Wilcoxon test. Relationships between the expression level of MALAT1, patient clinicopathological parameters and disease-free survival (DFS) and overall survival (OS) were analysed using the univariate Kaplan-Meier method and the multivariate COX regression model. Results: The MALAT1 levels in cancerous tissues were 2.26 times higher than those measured in noncancerous tissues, and this difference was statistically significant (P = 0.0004). Based on their expression level of MALAT1, the patients were divided into a high MALAT1 expression group (n = 73) and a low expression group (n = 73). Patients with tumours harbouring higher expression of MALAT1 showed a significantly worse prognosis with a hazard ratio (HR) of 2.863 (95% CI, 1.659 to 4.943; P < 0.001) for DFS and 3.968 (95% CI, 1.665 to 9.456; P = 0.002) for OS. Furthermore, patients with perineural invasion demonstrated significantly worse DFS (HR = 3.459, 95% CI 2.008 to 5.957; P < 0.001) and OS (HR = 3.750, 95% CI 1.743 to 8.069; P = 0.001) than those without perineural invasion. Multivariate analyses indicated that MALAT1 expression and perineural invasion were two independent prognostic risk factors for patients with CRC. Conclusion: The expression of MALAT1 is upregulated in CRC tissues, and a higher expression level of MALAT1 might serve as a negative prognostic marker in stage II/III CRC patients. PMID:25031737

  4. Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Corynebacterium glutamicum

    PubMed Central

    Molenaar, Douwe; van der Rest, Michel E.; Drysch, André; Yücel, Raif

    2000-01-01

    Like many other bacteria, Corynebacterium glutamicum possesses two types of l-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure. PMID:11092846

  5. Evidence That a Malate/Inorganic Phosphate Exchange Translocator Imports Carbon across the Leucoplast Envelope for Fatty Acid Synthesis in Developing Castor Seed Endosperm.

    PubMed Central

    Eastmond, P. J.; Dennis, D. T.; Rawsthorne, S.

    1997-01-01

    In this study we examined the processes by which malate and pyruvate are taken up across the leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids. PMID:12223747

  6. 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. PMID:27284052

  7. Evaluating the potential role of pomegranate peel in aluminum-induced oxidative stress and histopathological alterations in brain of female rats.

    PubMed

    Abdel Moneim, Ahmed E

    2012-12-01

    Studies have shown that pomegranate, Punica granatum Linn. (Lythraceae), has remarkable biological and medicinal properties. However, the effects of pomegranate peel methanolic extract (PPME) on the aluminum-induced oxidative stress and histopathological change have not been reported yet. To determine the effect of PPME (200 mg/kg bwt) on the aluminum chloride (AlCl₃; 34 mg/kg bwt)-induced neurotoxicity, aluminum accumulation in brain and oxidant/antioxidant status were determined. The change of brain structure was investigated with hematoxylin and eosin, and anti-apoptosis effects of PPME were analyzed by immunohistochemistry. The present study showed an indication of carcinogenicity in the AlCl₃-treated group representing an increase in tissue tumor markers such as tumor necrosis factor-α and angiogenin and inflammation by inducing an increase in prostaglandin E2 and prostaglandin F2α. PPME protected brain through decreasing the aluminum accumulation and stimulating antioxidant activities and anti-apoptotic proteins namely Bcl-2. Therefore, these results indicated that pomegranate peel methanolic extract could inhibit aluminum-induced oxidative stress and histopathological alternations in brain of female rats, and these effects may be related to anti-apoptotic and antioxidants activities. PMID:22945624

  8. Long noncoding RNA MALAT1 as a putative biomarker of lymph node metastasis: a meta-analysis

    PubMed Central

    Zhai, Hui; Chen, Qing-Jie; Chen, Bang-Dang; Yang, Yi-Ning; Ma, Yi-Tong; Li, Xiao-Mei; Liu, Fen; Yu, Zi-Xiang; Xiang, Yang; Liao, Wu; Lai, Hong-Mei

    2015-01-01

    Recent studies in cancer have demonstrated that cancerous tissues have a significantly higher MALAT1 level than in noncancerous tissues. Overexpression of MALAT1 is associated with susceptibility to lymph node metastasis. This meta-analysis collected all relevant articles and explored the association of MALAT1 expression levels with lymph node metastasis in patients with carcinoma. Literature collections were conducted by searching electronic databases PubMed, Cochrane Library, Web of Science (up to January 20, 2015). The odds ratio (OR) and its corresponding 95% confidence interval (CI) were calculated to assess the strength of the association by using RevMan5.1 software. A total of 573 patients from 5 studies were included in this meta-analysis. The results showed lymph node metastasis occurred more frequently in patients with high MALAT1 expression group than in patients with low MALAT1 expression group (OR = 2.64, 95% CI 1.06-6.56, P = 0.04 random-effects model). This meta-analysis demonstrated that overexpression of MALAT1 is significantly associated with lymph node metastasis in carcinoma patients. PMID:26221312

  9. L-Malate's Plasma and Excretion Profile in the Treatment of Moderate and Severe Hemorrhagic Shock in Rats

    PubMed Central

    Himmen, Stephan; Mueller, Friederike; Rohrig, Ricarda; Roehrborn, Friederike; Teloh, Johanna K.; de Groot, Herbert

    2016-01-01

    Introduction. Malate is a standard component in fluid therapy within a wide range of medical applications. To date, there are insufficient data regarding its plasma distribution, renal excretion, and metabolism after infusion. This study aimed to investigate these three aspects in a rat model of moderate and severe hemorrhagic shock (HS). Methods. Male Wistar rats were subjected to HS by dropping the mean arterial blood pressure to 25–30 mmHg (severe) and 40–45 mmHg (moderate), respectively, for 60 minutes. Subsequently, reperfusion with Ringer-saline or a malate containing crystalloid solution (7 mM, 13.6 mM, and 21 mM, resp.) was performed within 30 minutes, followed by an observation period of 150 minutes. Results. In the present experiments, malate rapidly disappeared from the blood, while only 5% of the infused malate was renally excreted. In the resuscitation interval the urinary citrate and succinate amounts significantly increased compared to control. Conclusion. Malate's half-life is between 30 and 60 minutes in both, moderate and severe HS. Thus, even under traumatic conditions malate seems to be subjected to rapid metabolism with participation of the kidneys. PMID:27403429

  10. Observation of cytoplasmic and vacuolar malate in maize root tips by sup 13 C-NMR spectroscopy. [Zea mays L

    SciTech Connect

    Chang, K.; Roberts, J.K.M. )

    1989-01-01

    The accumulation of malate by maize (Zea mays L.) root tips perfused with KH{sup 13}CO{sub 3} was followed by {sup 13}C nuclear magnetic resonance spectroscopy. In vivo nuclear magnetic resonance spectra contained distinct signals from two pools of malate in maize root tips, one at a pH {approximately}5.3 (assigned to the vacuole) and one at a pH > 6.5 (assigned to the cytoplasm). The ratio of cytoplasmic to vacuolar malate was lower in 12 millimeter long root tips than in 2 millimeter root tips. The relatively broad width of the signals from C1- and C4-labeled vacuolar malate indicated heterogeneity in vacuolar pH. During the 3 hour KH{sup 13}CO{sub 3} treatment, {sup 13}C-malate accumulated first primarily in the cytoplasm, increasing to a fairly constant level of {approximately}6 millimolar by 1 hour. After a lag, vacuolar malate increased throughout the experiment.

  11. Coordinate expression of transcriptionally regulated isocitrate lyase and malate synthase genes in Brassica napus L.

    PubMed Central

    Comai, L; Dietrich, R A; Maslyar, D J; Baden, C S; Harada, J J

    1989-01-01

    We have analyzed the temporal and spatial expression of genes encoding the glycoxylate cycle enzymes isocitrate lyase and malate synthase in Brassica napus L. to determine whether they are coordinately expressed. Both enzymes participate in reactions associated with lipid mobilization in oilseed plant seedlings and are sequestered in a specialized organelle, the glyoxysome. We have identified an isocitrate lyase cDNA clone containing the complete protein coding region. RNA blot and in situ hybridization studies with isocitrate lyase and malate synthase cDNA clones from B. napus showed that the genes exhibit similar expression patterns. The mRNAs begin to accumulate during late embryogeny, reach maximal levels in seedling cotyledons, are not detected at significant amounts in leaves, and are distributed similarly in cotyledons and axes of seedlings. Furthermore, transcription studies with isolated nuclei indicate that the genes are controlled primarily although not exclusively at the transcriptional level. We conclude that glyoxysome biogenesis is regulated in part through the coordinate expression of isocitrate lyase and malate synthase genes. PMID:2535504

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

    PubMed

    Oh, Se Jeong; Gu, Dong Ryun; Jin, Su Hyun; Park, Keun Ha; Lee, Seoung Hoon

    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 reduced 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. PMID:27179783

  13. Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

    PubMed

    Gharib, Ghazaleh; Rashid, Naeem; Bashir, Qamar; Gardner, Qura-Tul Ann Afza; Akhtar, Muhammad; Imanaka, Tadayuki

    2016-01-01

    Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water. PMID:26507956

  14. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

    PubMed

    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle. PMID:198130

  15. Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum.

    PubMed

    Tesfaye, M; Temple, S J; Allan, D L; Vance, C P; Samac, D A

    2001-12-01

    Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity. PMID:11743127

  16. Characterization of the immunogenicity and pathogenicity of malate dehydrogenase in Brucella abortus.

    PubMed

    Han, Xiangan; Tong, Yongliang; Tian, Mingxing; Sun, Xiaoqing; Wang, Shaohui; Ding, Chan; Yu, Shengqing

    2014-07-01

    Brucella abortus is a gram-negative, facultative intracellular pathogen that causes brucellosis, a chronic zoonotic disease resulting in abortion in pregnant cattle and undulant fever in humans. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, plays important metabolic roles in aerobic energy producing pathways and in malate shuttle. In this study, the MDH-encoding gene for malate dehydrogenase mdh of B. abortus S2308 was cloned, sequenced and expressed. Western blot analysis demonstrated that MDH is an immunogenic membrane-associated protein. In addition, recombinant MDH showed sero-reactivity with 30 individual bovine B. abortus-positive sera by enzyme-linked immunosorbent assay, indicates that MDH may be used as a candidate marker for sero-diagnosis of brucellosis. Furthermore, MDH exhibits fibronectin and plasminogen-binding ability in immunoblotting assay. Inhibition assays on HeLa cells demonstrated that rabbit anti-serum against MDH significantly reduced both bacterial adherence and invasion abilities (p < 0.05), suggesting that MDH play a role in B. abortus colonization. Our results indicated that MDH is not only an immunogenic protein, but is also related to bacterial pathogenesis and may act as a new virulent factor, which will benefit for further understanding the MDH's roles in B. abortus metabolism, pathogenesis and immunity. PMID:24609497

  17. Final report on the safety assessment of Malic Acid and Sodium Malate.

    PubMed

    Fiume, Z

    2001-01-01

    Malic Acid functions in cosmetic formulations as a pH adjuster, and Sodium Malate functions as a skin conditioning agent-humectant. Malic Acid is reportedly used in almost 50 cosmetic formulations across a range of product types at low concentrations, whereas Sodium Malate is used in only one. As a pH adjuster, Malic Acid is used at low concentrations. One commercial method of preparing Malic Acid is hydration of fumaric acid or maleic acid, and then purified to limit the amount of the starting material present. Because Malic Acid is a component of the Kreb's cycle, another method is fermentation. Malic Acid was relatively nontoxic in acute toxicity studies using animals. In a chronic oral study, feeding Malic Acid to rats resulted only in weight gain changes and changes in feed consumption. Malic Acid did not cause reproductive toxicity in mice, rats, or rabbits. Malic Acid was a moderate to strong skin irritatant in animal tests, and was a strong ocular irritant. Malic Acid was not mutagenic across a range of genotoxicity tests. Malic Acid was irritating in clinical tests, with less irritation seen as pH of the applied material increased. Patients patch tested with Malic Acid, placed on a diet that avoided foods containing Malic or citric acid, and then challenged with a diet high in Malic and citric acid had both immediate urticarial and delayed contact dermatitis reactions. These data were considered sufficient to determine that Malic Acid and Sodium Malate would be safe at the low concentrations at which these ingredients would be used to adjust pH (even though Sodium Malate is not currently used for that purpose). The data, however, were insufficient to determine the safety of these ingredients when used in cosmetics as other than pH adjusters and specifically, the data are insufficient to determine the safety of Sodium Malate when used as a skin conditioning agent-humectant. The types of data required for the Expert Panel to determine the safety of Sodium

  18. Identification of Pseudomonas fluorescens Chemotaxis Sensory Proteins for Malate, Succinate, and Fumarate, and Their Involvement in Root Colonization

    PubMed Central

    Oku, Shota; Komatsu, Ayaka; Nakashimada, Yutaka; Tajima, Takahisa; Kato, Junichi

    2014-01-01

    Pseudomonas fluorescens Pf0-1 exhibited chemotactic responses to l-malate, succinate, and fumarate. We constructed a plasmid library of 37 methyl-accepting chemotaxis protein (MCP) genes of P. fluorescens Pf0-1. To identify a MCP for l-malate, the plasmid library was screened using the PA2652 mutant of Pseudomonas aeruginosa PAO1, a mutant defective in chemotaxis to l-malate. The introduction of Pfl01_0728 and Pfl01_3768 genes restored the ability of the PA2652 mutant to respond to l-malate. The Pfl01_0728 and Pfl01_3768 double mutant of P. fluorescens Pf0-1 showed no response to l-malate or succinate, while the Pfl01_0728 single mutant did not respond to fumarate. These results indicated that Pfl01_0728 and Pfl01_3768 were the major MCPs for l-malate and succinate, and Pfl01_0728 was also a major MCP for fumarate. The Pfl01_0728 and Pfl01_3768 double mutant unexpectedly exhibited stronger responses toward the tomato root exudate and amino acids such as proline, asparagine, methionine, and phenylalanine than those of the wild-type strain. The ctaA, ctaB, ctaC (genes of the major MCPs for amino acids), Pfl01_0728, and Pfl01_3768 quintuple mutant of P. fluorescens Pf0-1 was less competitive than the ctaA ctaB ctaC triple mutant in competitive root colonization, suggesting that chemotaxis to l-malate, succinate, and/or fumarate was involved in tomato root colonization by P. fluorescens Pf0-1. PMID:25491753

  19. Long Non Coding RNA MALAT1 Promotes Tumor Growth and Metastasis by inducing Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma

    PubMed Central

    Zhou, Xuan; Liu, Su; Cai, Guoshuai; Kong, Lingping; Zhang, Tingting; Ren, Yu; Wu, Yansheng; Mei, Mei; Zhang, Lun; Wang, Xudong

    2015-01-01

    The prognosis of advanced oral squamous cell carcinoma (OSCC) patients remains dismal, and a better understanding of the underlying mechanisms is critical for identifying effective targets with therapeutic potential to improve the survival of patients with OSCC. This study aims to clarify the clinical and biological significance of metastasis-associated long non-coding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in OSCC. We found that MALAT1 is overexpressed in OSCC tissues compared to normal oral mucosa by real-time PCR. MALAT1 served as a new prognostic factor in OSCC patients. When knockdown by small interfering RNA (siRNA) in OSCC cell lines TSCCA and Tca8113, MALAT1 was shown to be required for maintaining epithelial-mesenchymal transition (EMT) mediated cell migration and invasion. Western blot and immunofluorescence staining showed that MALAT1 knockdown significantly suppressed N-cadherin and Vimentin expression but induced E-cadherin expression in vitro. Meanwhile, both nucleus and cytoplasm levels of β-catenin and NF-κB were attenuated, while elevated MALAT1 level triggered the expression of β-catenin and NF-κB. More importantly, targeting MALAT1 inhibited TSCCA cell-induced xenograft tumor growth in vivo. Therefore, these findings provide mechanistic insight into the role of MALAT1 in regulating OSCC metastasis, suggesting that MALAT1 is an important prognostic factor and therapeutic target for OSCC. PMID:26522444

  20. Downregulation of lncRNA-MALAT1 Affects Proliferation and the Expression of Stemness Markers in Glioma Stem Cell Line SHG139S.

    PubMed

    Han, Yong; Zhou, Liang; Wu, Tingfeng; Huang, Yulun; Cheng, Zhe; Li, Xuetao; Sun, Ting; Zhou, Youxin; Du, Ziwei

    2016-10-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is among the most abundant and highly conserved lncRNAs, which has been detected in a wide variety of human tumors, including gastric cancer, gallbladder cancer, and so on. Previous research has showed that MALAT1 can activate LTBP3 gene in mesenchymal stem cells. However, the specific roles of MALAT1 in glioma stem cells (GSCs) remain unclear. In this study, we aimed to identify the effects of MALAT1 on proliferation and the expression of stemness markers on glioma stem cell line SHG139S. Our results showed that downregulation of MALAT1 suppressed the expression of Sox2 and Nestin which are related to stemness, while downregulation of MALAT1 promoted the proliferation in SHG139S. Further research on the underlying mechanism showed that the effects of MALAT1 downregulation on SHG139S were through regulating ERK/MAPk signaling activity. And we also found that downregulation of MALAT1 could activate ERK/MAPK signaling and promoted proliferation in SHG139 cells. These findings show that MALAT1 plays an important role in regulating the expression of stemness markers and proliferation of SHG139S, and provide a new research direction to target the progression of GSCs. PMID:26649728

  1. Plasma long non-coding RNA MALAT1 is associated with distant metastasis in patients with epithelial ovarian cancer

    PubMed Central

    Chen, Qingjuan; Su, Yongyong; He, Xiaopeng; Zhao, Weian; Wu, Caixia; Zhang, Weibo; Si, Xiaomin; Dong, Bingwei; Zhao, Lianying; Gao, Yufang; Yang, Xiaowen; Chen, Jianhui; Lu, Jian; Qiao, Ximin; Zhang, Yuchen

    2016-01-01

    Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a newly identified metastasis-associated long non-coding RNA. In a previous study, it was identified that plasma levels of MALAT1 were significantly increased in gastric cancer patients with metastasis compared with gastric cancer patients without metastasis and healthy control individuals. However, it is unclear whether plasma levels of MALAT1 may act as a biomarker for evaluating the development of metastasis in epithelial ovarian cancer (EOC). In the present study, groups that consisted of 47 patients with EOC and metastasis (EOC/DM), 47 patients with EOC without metastasis (EOC/NDM), and 47 healthy control (HC) individuals were established. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the level of plasma MALAT1 in these groups. The results showed that levels of plasma MALAT1 were significantly increased in the EOC/DM group compared with the EOC/NDM and HC groups (P<0.001). Receiver operating characteristic (ROC) analysis indicated that plasma MALAT1 yielded an area under the curve (AUC) of 0.820 [95% confidence interval (CI), 0.734–0.905; P<0.001], distinguishing between EOC/DM and EOC/NDM. ROC analysis also yielded an AUC of 0.884 (95% CI, 0.820–0.949; P<0.001), with 89.4% sensitivity and 72.3% specificity for distinguishing between EOC/DM and HC. Furthermore, multivariate analysis indicated that overexpression of MALAT1, differentiation (poor), tumor-node-metastasis stage (IV), lymph node metastasis (N3), peritoneal invasion (present) and higher serum carbohydrate antigen 125 levels were independent predictors of survival (hazard ratio, 3.322; P=0.028) in patients with EOC. Kaplan-Meier analysis revealed that patients with increased MALAT1 expression had a poorer disease-free survival time. In conclusion, the levels of plasma MALAT1 may act as a valuable biomarker for the diagnosis of metastasis. PMID:27446438

  2. The glcB locus of Rhizobium leguminosarum VF39 encodes an arabinose-inducible malate synthase.

    PubMed

    García-de los Santos, Alejandro; Morales, Alejandro; Baldomá, Laura; Clark, Scott R D; Brom, Susana; Yost, Christopher K; Hernández-Lucas, Ismael; Aguilar, Juan; Hynes, Michael F

    2002-10-01

    In the course of a study conducted to isolate genes upregulated by plant cell wall sugars, we identified an arabinose-inducible locus from a transcriptional fusion library of Rhizobium leguminosarum VF39, carrying random insertions of the lacZ transposon Tn5B22. Sequence analysis of the locus disrupted by the transposon revealed a high similarity to uncharacterized malate synthase G genes from Sinorhizobium meliloti, Agrobacterium tumefaciens, and Mesorhizobium loti. This enzyme catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA and is thought to be a component of the glyoxylate cycle, which allows microorganisms to grow on two carbon compounds. Enzyme assays showed that a functional malate synthase is encoded in the glcB gene of R. leguminosarum and that its expression is induced by arabinose, glycolate, and glyoxylate. An Escherichia coli aceB glcB mutant, complemented with the R. leguminosarum PCR-amplified gene, recovered malate synthase activity. A very similar genome organization of the loci containing malate synthase and flanking genes was observed in R. leguminosarum, S. meliloti, and A. tumefaciens. Pea plants inoculated with the glcB mutant or the wild-type strain showed no significant differences in nitrogen fixation. This is the first report regarding the characterization of a mutant in one of the glyoxylate cycle enzymes in the rhizobia. PMID:12489782

  3. Maintenance carbon cycle in crassulacean Acid metabolism plant leaves : source and compartmentation of carbon for nocturnal malate synthesis.

    PubMed

    Kenyon, W H; Severson, R F; Black, C C

    1985-01-01

    The reciprocal relationship between diurnal changes in organic acid and storage carbohydrate was examined in the leaves of three Crassulacean acid metabolism plants. It was found that depletion of leaf hexoses at night was sufficient to account quantitatively for increase in malate in Ananas comosus but not in Sedum telephium or Kalanchoë daigremontiana. Fructose and to a lesser extent glucose underwent the largest changes. Glucose levels in S. telephium leaves oscillated diurnally but were not reciprocally related to malate fluctuations.Analysis of isolated protoplasts and vacuoles from leaves of A. comosus and S. telephium revealed that vacuoles contain a large percentage (>50%) of the protoplast glucose, fructose and malate, citrate, isocitrate, ascorbate and succinate. Sucrose, a major constituent of intact leaves, was not detectable or was at extremely low levels in protoplasts and vacuoles from both plants.In isolated vacuoles from both A. comosus and S. telephium, hexose levels decreased at night at the same time malate increased. Only in A. comosus, however, could hexose metabolism account for a significant amount of the nocturnal increase in malate. We conclude that, in A. comosus, soluble sugars are part of the daily maintenance carbon cycle and that the vacuole plays a dynamic role in the diurnal carbon assimilation cycle of this Crassulacean acid metabolism plant. PMID:16664005

  4. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate.

    PubMed

    Laroute, Valérie; Yasaro, Chonthicha; Narin, Waranya; Mazzoli, Roberto; Pessione, Enrica; Cocaign-Bousquet, Muriel; Loubière, Pascal

    2016-01-01

    Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes. PMID:27458444

  5. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate

    PubMed Central

    Laroute, Valérie; Yasaro, Chonthicha; Narin, Waranya; Mazzoli, Roberto; Pessione, Enrica; Cocaign-Bousquet, Muriel; Loubière, Pascal

    2016-01-01

    Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes. PMID:27458444

  6. Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test

    SciTech Connect

    Clark, J.D.; O'Keefe, S.J.; Knowles, J.R.

    1988-08-09

    Although aldolase-catalyzed condensations proceed by stepwise mechanisms via the intermediacy of nucleophilic enol(ate)s or enamines, the mechanisms of those enzymes that catalyze Claisen-type condensations are unclear. The reaction pathway followed by an enzyme from this second group, malate synthase, has been studied by the double-isotope fractionation method to determine whether the reaction is stepwise or concerted. In agreement with earlier work, a deuterium kinetic isotope effect /sup D/(V/K) of 1.3 +/- 0.1 has been found when (/sup 2/H/sub 3/)acetyl-CoA is the substrate. The /sup 13/C isotope effect at the aldehydic carbon of glyoxylate has also been measured. For this determination, the malate product was quantitatively transformed into a new sample of malate having the carbon of interest at C-4. This material was decarboxylated by malic enzyme to produce the appropriate CO/sub 2/ for isotope ratio mass spectrometric analysis. The /sup 13/C isotope effect with (/sup 1/H/sub 3/)acetyl-CoA is 1.0037 +/- 0.0004. By use of the known values of the intermolecular and intramolecular deuterium effects and of /sup 13/(V/K)/sub H/, the value of the /sup 13/C isotope effect when deuteriated (/sup 2/H/sub 3/)acetyl-CoA is the substrate can be predicted for three possible mechanisms. The results show clearly that the two salient characteristics of enzymes that catalyze Claisen-like condensations, namely, the absence of enzyme-catalyzed proton exchange with solvent and the inversion of the configuration at the nucleophilic center, which had been suggestive of a concerted pathway, are not mechanistically diagnostic.

  7. Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein.

    PubMed

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon; Kim, Suk

    2016-03-01

    The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis. PMID:27051349

  8. Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein

    PubMed Central

    Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Arayan, Lauren Togonon

    2016-01-01

    The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis. PMID:27051349

  9. Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit.

    PubMed

    Vermouth, N T; Carriazo, C S; Ponce, R H; Blanco, A

    1986-01-01

    Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda. PMID:3956158

  10. Effects of supplemental citrulline malate ingestion during repeated bouts of lower-body exercise in advanced weightlifters.

    PubMed

    Wax, Benjamin; Kavazis, Andreas N; Weldon, Kevin; Sperlak, Joseph

    2015-03-01

    The purpose of this investigation was to test the efficacy of citrulline malate supplementation on exercise performance, blood lactate, heart rate, and blood pressure during lower-body dynamic resistance exercise. We hypothesized that citrulline malate ingestion before performing submaximal repeated bouts of multiple lower-body resistance exercises would improve performance. Twelve advanced resistance-trained male subjects participated in a randomized, counterbalanced, double-blind study. Subjects were randomly assigned to placebo (PL) or citrulline malate (8 g) groups and then performed repeated bouts of multiple lower-body resistance exercise. Specifically, subjects performed 5 sequential sets (60% 1 repetition maximum) to failure on the leg press, hack squat, and leg extension machines. Blood lactate, heart rate, systolic blood pressure, and diastolic blood pressure were determined before and after exercise. The exercise protocol resulted in sequential significant (p ≤ 0.05) decrease in the number of repetitions in all 3 exercises. However, subjects in the citrulline malate group performed significantly (p ≤ 0.05) higher number of repetitions during all 3 exercises compared with PL group. Blood lactate and heart rate were significantly increased (p ≤ 0.05) after exercise compared with before exercise but were not significantly different between citrulline malate and PL (p > 0.05). No significant (p > 0.05) differences were detected for blood pressure measurements. In conclusion, our results suggest that citrulline malate supplementation may be beneficial in improving exercise performance during lower-body multiple-bout resistance exercise in advanced resistance-trained men. PMID:25226311

  11. Tumor-suppressive function of long noncoding RNA MALAT1 in glioma cells by downregulation of MMP2 and inactivation of ERK/MAPK signaling

    PubMed Central

    Han, Y; Wu, Z; Wu, T; Huang, Y; Cheng, Z; Li, X; Sun, T; Xie, X; Zhou, Y; Du, Z

    2016-01-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a type of long noncoding RNA. It is associated with metastasis and is a favorable prognostic factor for lung cancer. Recent studies have shown that MALAT1 plays an important role in other malignancies. But, little is known about the role of MALAT1 in glioma. In this study, quantitative reverse transcription PCR (qRT-PCR) was used to demonstrate that the expression of MALAT1 was lower than that in normal brain tissues. Stable RNA interference-mediated knockdown of MALAT1 in human glioma cell lines (U87 and U251) significantly promoted the invasion and proliferation of the glioma cells by in vitro assays. Conversely, overexpression of MALAT1 caused significant reduction in cell proliferation and invasion in vitro, and tumorigenicity in both subcutaneous and intracranial human glioma xenograft models. Furthermore, MALAT1-mediated tumor suppression in glioma cells may be via reduction of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling activity and expression of matrix metalloproteinase 2 (MMP2). In conclusion, overall data demonstrated the tumor-suppressive role of MALAT1 in glioma by attenuating ERK/MAPK-mediated growth and MMP2-mediated invasiveness. PMID:26938295

  12. miR-124 downregulation leads to breast cancer progression via LncRNA-MALAT1 regulation and CDK4/E2F1 signal activation

    PubMed Central

    Feng, Tongbao; Shao, Fang; Wu, Qiyong; Zhang, Xiaohang; Xu, Dongqin; Qian, Keqing; Xie, Yewen; Wang, Shizhong; Xu, Ning; Wang, Yong; Qi, Chunjian

    2016-01-01

    The long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been recently shown to be dysregulated in several cancers. However, the mechanisms underlying the role of MALAT1 in breast cancer remain unclear. Herein, we showed that MALAT1 was aberrantly increased in breast cancer tissues and cells. MALAT1-siRNA inhibited breast cancer cell proliferation and cell cycle progression in vitro and in vivo. Furthermore, MALAT1 acted as an endogenous potent regulator by directly binding to miR-124 and down-regulating miR-124 expression. In addition, MALAT1 reversed the inhibitory effect of miR-124 on breast cancer proliferation and was involved in the cyclin-dependent kinase 4 (CDK4) expression. Taken together, our data highlight the pivotal role of MALAT1 in breast cancer tumorigenesis. Moreover, the present study elucidated the MALAT1-miR-124-CDK4/E2F1 signaling pathway in breast cancer, which might provide a new approach for tackling breast cancer. PMID:26918449

  13. Evaluation of 90-day Repeated Dose Oral Toxicity, Glycometabolism, Learning and Memory Ability, and Related Enzyme of Chromium Malate Supplementation in Sprague-Dawley Rats.

    PubMed

    Feng, Weiwei; Wu, Huiyu; Li, Qian; Zhou, Zhaoxiang; Chen, Yao; Zhao, Ting; Feng, Yun; Mao, Guanghua; Li, Fang; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the 90-day oral toxicity of chromium malate in Sprague-Dawley rats. The present study inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, lipid metabolism, and learning and memory ability in metabolically healthy Sprague-Dawley rats. The results showed that all rats survived and pathological, toxic, feces, and urine changes were not observed. Chromium malate did not cause measurable damage on liver, brain, and kidney. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of normal rats in chromium malate groups had no significant change when compared with control group and chromium picolinate group under physiologically relevant conditions. The serum and organ content of Cr in chromium malate groups had no significant change compared with control group. No significant changes were found in morris water maze test and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and true choline esterase (TChE) activity. The results indicated that supplementation with chromium malate did not cause measurable toxicity and has no obvious effect on glycometabolism and related enzymes, learning and memory ability, and related enzymes and lipid metabolism of female and male rats. The results of this study suggest that chromium malate is safe for human consumption. PMID:25900579

  14. 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. PMID:26983942

  15. Long non-coding RNA MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation in colorectal cancer cells

    PubMed Central

    Xie, Lin-Ying; Huang, Yu-qi; Liu, Yan-Ping; Xiao, Li-Wei; Li, Sheng-Nan; Zhu, Hui-Fang; Li, Zu-Guo; Kan, Heping

    2016-01-01

    Our earlier findings indicate that the long non-coding RNA MALAT1 promotes colorectal cancer (CRC) cell proliferation, invasion and metastasis in vitro and in vivo by increasing expression of AKAP-9. In the present study, we investigated the molecular mechanism by which MALAT1 enhances AKAP9 expression in CRC SW480 cells. We found that MALAT1 interacts with both SRPK1 and SRSF1. MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation. Following MALAT1 knockdown, overexpression of SRPK1 was sufficient to restore SRSF1 phosphorylation and AKAP-9 expression to a level that promoted cell proliferation, invasion and migration in vitro. Conversely, SRPK1 knockdown after overexpression of MALAT1 in SW480 cells diminished SRSF1 phosphorylation and AKAP-9 expression and suppressed cell proliferation, invasion and migration in vitro. These findings suggest MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation in CRC cells. These results reveal a novel molecular mechanism by which MALAT1 regulates AKAP-9 expression in CRC cells. PMID:26887056

  16. Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase

    PubMed Central

    Liu, Yan; Asnani, Aarti; Zou, Lin; Bentley, Victoria L.; Yu, Min; Wang, You; Dellaire, Graham; Sarkar, Kumar S.; Dai, Matthew; Chen, Howard H.; Sosnovik, David E.; Shin, Jordan T.; Haber, Daniel A.; Berman, Jason N.; Chao, Wei; Peterson, Randall T.

    2015-01-01

    Doxorubicin is a highly effective anti-cancer chemotherapy agent, but its usage is limited by its cardiotoxicity. To develop a drug that prevents the cardiac toxicity of doxorubicin while preserving its anti-tumor potency, we established a doxorubicin-induced cardiomyopathy model in zebrafish that recapitulated the cardiomyocyte apoptosis and contractility decline observed in patients. Using this model, we screened 3000 compounds and discovered that visnagin (VIS) and diphenylurea (DPU) rescue cardiac performance and circulatory defects caused by doxorubicin treatment in zebrafish. VIS and DPU reduced doxorubicin-induced apoptosis in cultured cardiomyocytes and in vivo in zebrafish and mouse hearts. Furthermore, VIS treatment improved cardiac contractility in doxorubicin-treated mice. Importantly, VIS and DPU caused no reduction in the chemotherapeutic efficacy of doxorubicin in several cultured tumor lines or in zebrafish and mouse xenograft models. Using affinity chromatography, we discovered that VIS binds to mitochondrial malate dehydrogenase (MDH2), one of the key enzymes in the tricarboxylic acid cycle. As with VIS, treatment with the MDH2 inhibitors mebendazole, thyroxine, and iodine prevented doxorubicin cardiotoxicity, as did treatment with malate itself, suggesting that modulation of MDH2 activity is responsible for VIS’s cardioprotective effects. Taken together, this study identified VIS and DPU as potent cardioprotective compounds and implicates MDH2 as a previously undescribed, druggable target for doxorubicin-induced cardiomyopathy. PMID:25504881

  17. Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase.

    PubMed

    Liu, Yan; Asnani, Aarti; Zou, Lin; Bentley, Victoria L; Yu, Min; Wang, You; Dellaire, Graham; Sarkar, Kumar S; Dai, Matthew; Chen, Howard H; Sosnovik, David E; Shin, Jordan T; Haber, Daniel A; Berman, Jason N; Chao, Wei; Peterson, Randall T

    2014-12-10

    Doxorubicin is a highly effective anticancer chemotherapy agent, but its use is limited by its cardiotoxicity. To develop a drug that prevents this toxicity, we established a doxorubicin-induced cardiomyopathy model in zebrafish that recapitulates the cardiomyocyte apoptosis and contractility decline observed in patients. Using this model, we screened 3000 compounds and found that visnagin (VIS) and diphenylurea (DPU) rescue the cardiac performance and circulatory defects caused by doxorubicin in zebrafish. VIS and DPU reduced doxorubicin-induced apoptosis in cultured cardiomyocytes and in vivo in zebrafish and mouse hearts. VIS treatment improved cardiac contractility in doxorubicin-treated mice. Further, VIS and DPU did not reduce the chemotherapeutic efficacy of doxorubicin in several cultured tumor lines or in zebrafish and mouse xenograft models. Using affinity chromatography, we found that VIS binds to mitochondrial malate dehydrogenase (MDH2), a key enzyme in the tricarboxylic acid cycle. As with VIS, treatment with the MDH2 inhibitors mebendazole, thyroxine, and iodine prevented doxorubicin cardiotoxicity, as did treatment with malate itself, suggesting that modulation of MDH2 activity is responsible for VIS' cardioprotective effects. Thus, VIS and DPU are potent cardioprotective compounds, and MDH2 is a previously undescribed, druggable target for doxorubicin-induced cardiomyopathy. PMID:25504881

  18. Phosphorylation of glyoxysomal malate synthase from castor oil seed endosperm and cucumber cotyledon

    SciTech Connect

    Yang, Y.P; Randall, D.D. )

    1989-04-01

    Glyoxysomal malate synthase (MS) was purified to apparent homogeneity from 3-d germinating castor oil seed endosperm by a relatively simple procedure including two sucrose density gradient centrifugations. Antibodies raised to the caster oil seed MS crossreacted with MS from cucumber cotyledon. MS was phosphorylated in both tissues in an MgATP dependent reaction. The phosphorylation pattern was similar for both enzymes and both enzymes were inhibited by NaF, NaMo, (NH{sub 4})SO{sub 4}, glyoxylate and high concentration of MgCl{sub 2} (60 mM), but was not inhibited by NaCl and malate. Further characterization of the phosphorylation of MS from castor oil seed endosperms showed that the 5S form of MS is the form which is labelled by {sup 32}P. The addition of exogenous alkaline phosphatase to MS not only decreased enzyme activity, but could also dephosphorylate phospho-MS. The relationship between dephosphorylation of MS and the decrease of MS activity is currently under investigation.

  19. Optimized extraction of calcium malate from eggshell treated by PEF and an absorption assessment in vitro.

    PubMed

    Lin, Songyi; Wang, Liyan; Jones, Gregory; Trang, Hung; Yin, Yongguang; Liu, Jingbo

    2012-06-01

    Under optimized pulsed electric field (PEF) treatment for production of eggshell calcium malate (ESCM) by one-factor-at-a-time test and ternary quadratic regression orthogonal combination design (TQROCD), an absorption assessment of ESCM treated by the best conditions of PEF were performed in male mice with apparent calcium absorption rate (ACAR), serum alkalinity phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), serum calcium and serum phosphorus, length of femurs and skeletal calcium content were studied. The highest dissoluble calcium malate content (7.075 mg/mL) was obtained with the 6.0% malic acid, the electric field intensity of 20 kV/cm, and pulse duration of 24 μs. In vitro, ESCM prepared by the best conditions of PEF at doses of 133.0 mg kg(-1) d(-1) for 70 d not only significantly improve the ALP activity, the femur length and calcium content of bone of the mice (P<0.05) but also decreased the levels of TRAP (P<0.05). The ratio of calcium and phosphorus was in the normal range. PEF could be taken as a highly effective, environmentally friendly and energy-saving method for preparation of ESCM, which treated by PEF could promote the absorption of calcium in vitro, extraordinary can promote bone development and a healthy bone. PMID:22469913

  20. Characterization of <0 1 0> directed ammonium malate single crystals grown by Sankaranarayanan-Ramasamy method

    NASA Astrophysics Data System (ADS)

    Senthil Murugan, G.; Perumal Ramasamy, R.; Ramasamy, P.; Bhagavannarayana, G.

    2011-08-01

    Ammonium malate, an organic nonlinear optical single crystal, has been grown to a dimension of 50 mm length and 20 mm diameter by uniaxial solution-crystallization method of Sankaranarayanan-Ramasamy (SR). The growth conditions and experimental details of the grown crystals have been presented. The crystalline perfection of the grown crystals has been evaluated by high-resolution X-ray diffraction technique and the results reveal that the crystal quality of SR grown crystal is better than the conventional method grown crystal. The growth features in SR grown crystal have been analyzed by chemical etching technique and well-defined triangular etch hillocks have been observed. The mechanical property of the grown crystal has been analyzed by Vickers microhardness method and it reveals that the grown crystal is moderately harder material. Ammonium malate crystal possesses lower cut off wavelength at 230 nm. It enables good optical transmission of the second harmonic generation of Nd:YAG laser and other applications in the blue-violet region. The second harmonic generation of the grown crystal has been confirmed by the Kurtz powder technique.

  1. Uptake of malate dehydrogenase into mitochondria in vitro. Some characteristics of the process.

    PubMed Central

    Passarella, S; Marra, E; Doonan, S; Quagliariello, E

    1983-01-01

    1. It was previously shown [Passarella, Marra, Doonan & Quagliariello (1980) Biochem. J. 192, 649-658] that, when mitochondrial malate dehydrogenase from rat liver is incubated with sulphite-loaded mitochondria from the same source, uptake of the enzyme occurs, as judged by a fluorimetric assay of intramitochondrial enzyme activity. Confirmation of sequestration of the enzyme inside the organelles is provided by its proteinase-resistance after uptake. 2. Enzyme uptake into mitochondria is inhibited by enzyme treatment with mersalyl at concentrations that do not affect its catalytic activity. 3. Enzyme uptake is energy-dependent, as shown by inhibition of the process by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and by antimycin. ATP and oligomycin, on the other hand, both stimulate the process, but stimulation by ATP is inhibited by oligomycin. These results suggest that uptake depends on maintenance of transmembrane ion gradient rather than direct ATP involvement. 4. Measurements of delta psi by means of the 'redistribution signal' probe safranine suggest no dependence of malate dehydrogenase uptake on membrane potential. 5. Comparison of the effects of the ionophores valinomycin, nonactin, gramicidin and nigericin shows that uptake depends on maintenance of a transmembrane pH gradient. PMID:6847644

  2. Polyethylene glycol-induced heteroassociation of malate dehydrogenase and citrate synthase

    SciTech Connect

    Merz, J.M.; Webster, T.A.; Appleman, J.R.; Manley, E.R.; Yu, H.A.; Datta, A.; Ackerson, B.J.; Spivey, H.O.

    1987-10-01

    Studies by dynamic and total intensity light scattering, ultracentrifugation, electron microscopy, and chemical crosslinking on solutions of the pig heart mitochondrial enzymes, malate dehydrogenase and citrate synthase (separately and together) demonstrate that polyethylene glycol induces very large homoassociations of each enzyme, and still larger heteroenzyme complexes between these two enzymes in the solution phase. Specificity of this heteroassociation is indicated by the facts that heteroassociations with bovine serum albumin were not observed for either the mitochondrial dehydrogenase or the synthase or between cytosolic malate dehydrogenase and citrate synthase. The weight fraction of the enzymes in the mitochondrial dehydrogenase-synthase associated particles in the solution phase was less than 0.03% with the dilute conditions used in the dynamic light scattering measurements. Neither palmitoyl-CoA nor other solution conditions tested significantly increased this weight fraction of associated enzymes in the solution phase. Because of the extremely low solubility of the associated species, however, the majority of the enzymes can be precipitated as the heteroenzyme complex. This precipitation is a classical first-order transition in spite of the large particle sizes and broad size distribution. Ionic effects on the solubility of the heteroenzyme complex appear to be of general electrostatic nature. Polyethylene glycol was found to be more potent in precipitating this complex than dextrans, polyvinylpyrrolidones, ficoll, and beta-lactoglobulin.

  3. 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 vivo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 g...

  4. 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 nutrition.

    PubMed

    Lüttge, U; Pfeifer, T; Fischer-Schliebs, E; Ratajczak, R

    2000-11-01

    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, Deltamu(H+), 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, J(mal)(rel), 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. J(mal)(rel) 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 NO(3)(-) 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 J(mal)(rel). It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO(3)(-) nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins. PMID:11080309

  5. Resveratrol inhibits invasion and metastasis of colorectal cancer cells via MALAT1 mediated Wnt/β-catenin signal pathway.

    PubMed

    Ji, Qing; Liu, Xuan; Fu, Xiaoling; Zhang, Long; Sui, Hua; Zhou, Lihong; Sun, Jian; Cai, Jianfeng; Qin, Jianmin; Ren, Jianlin; Li, Qi

    2013-01-01

    Resveratrol, extracted from Chinese herbal medicine Polygonum cuspidatum, is known to inhibit invasion and metastasis of human colorectal cancer (CRC), in which long non-coding Metastasis Associated Lung Adenocarcinoma Transcript 1 (RNA-MALAT1) also plays an important role. Using MALAT1 lentiviral shRNA and over-expression constructs in CRC derived cell lines, LoVo and HCT116, we demonstrated that the anti-tumor effects of resveratrol on CRC are through inhibiting Wnt/β-catenin signaling, thus the expression of its target genes such as c-Myc, MMP-7, as well as the expression of MALAT1. In detail, resveratrol down-regulates MALAT1, resulting in decreased nuclear localization of β-catenin thus attenuated Wnt/β-catenin signaling, which leads to the inhibition of CRC invasion and metastasis. This finding of ours surely provides important pre-clinical evidence supporting future use of resveratrol in prevention and treatment of CRC. PMID:24244343

  6. Aluminum-activated citrate and malate transporters encoded by distinct Al tolerance genes function independently in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aluminum (Al) -activated malate and citrate exudation from roots plays an important role in conferring Al tolerance to many plant species. Here, we report on the identification and characterization of AtMATE, the gene encoding an Al-activated root citrate efflux transporter that functions in Arabid...

  7. SIRT3-dependent GOT2 acetylation status affects the malate-aspartate NADH shuttle activity and pancreatic tumor growth.

    PubMed

    Yang, Hui; Zhou, Lisha; Shi, Qian; Zhao, Yuzheng; Lin, Huaipeng; Zhang, Mengli; Zhao, Shimin; Yang, Yi; Ling, Zhi-Qiang; Guan, Kun-Liang; Xiong, Yue; Ye, Dan

    2015-04-15

    The malate-aspartate shuttle is indispensable for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis and to support rapid tumor cell growth. The malate-aspartate shuttle is operated by two pairs of enzymes that localize to the mitochondria and cytoplasm, glutamate oxaloacetate transaminases (GOT), and malate dehydrogenases (MDH). Here, we show that mitochondrial GOT2 is acetylated and that deacetylation depends on mitochondrial SIRT3. We have identified that acetylation occurs at three lysine residues, K159, K185, and K404 (3K), and enhances the association between GOT2 and MDH2. The GOT2 acetylation at these three residues promotes the net transfer of cytosolic NADH into mitochondria and changes the mitochondrial NADH/NAD(+) redox state to support ATP production. Additionally, GOT2 3K acetylation stimulates NADPH production to suppress ROS and to protect cells from oxidative damage. Moreover, GOT2 3K acetylation promotes pancreatic cell proliferation and tumor growth in vivo. Finally, we show that GOT2 K159 acetylation is increased in human pancreatic tumors, which correlates with reduced SIRT3 expression. Our study uncovers a previously unknown mechanism by which GOT2 acetylation stimulates the malate-aspartate NADH shuttle activity and oxidative protection. PMID:25755250

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium falciparum with Oligomeric Mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archea and other prokaryotes. Initial sequence a...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Fumarate permeation in normal and acidotic rat kidney mitochondria: fumarate/malate and fumarate/aspartate translocators.

    PubMed

    Atlante, A; Gagliardi, S; Passarella, S

    1998-02-24

    In order to gain some insight into the fate of fumarate synthesised in the cytosol in the purine nucleotide cycle and in amino acid catabolism, the capability of both rat kidney mitochondria and acidotic rat kidney mitochondria to take up either externally synthesised, via adenylsuccinate lyase, or added fumarate in exchange with intramitochondrial malate or aspartate was tested by means of both spectrophotometric and isotopic techniques. The appearance of either malate or aspartate caused by the presence of fumarate was revealed outside normal and acidotic mitochondria by using specific substrate detecting systems. Consistently, externally added fumarate was found to cause efflux of either [14C]-malate or [14C]-aspartate from loaded mitochondria. The occurrence in rat kidney mitochondria of two separate translocators, i.e., fumarate/malate and fumarate/aspartate carriers, is shown in the light of saturation kinetics and the different inhibitor sensitivity. The fumarate/aspartate antiporters found in normal and acidotic mitochondria appear to differ from each other. PMID:9500979

  12. The effect of five fasciolicides on malate dehydrogenase activity and mortality of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum.

    PubMed

    Probert, A J; Sharma, R K; Singh, K; Saxena, R

    1981-06-01

    The effect of oxyclozanide, hexachlorophene, nitroxynil, rafoxanide and diamphenethide on malate dehydrogenase activity of homogenates of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum was investigated. The ratio of oxaloacetate reduction to malate oxidation in homogenates of Fasciola gigantica, Fasciolopsis buski and P. explanatum was 4.5:1, 3.6:1 and 5.2:1 respectively. Oxyclozanide and rafoxanide at 10(-3) M inhibited enzyme activity by 100% in homogenates from all three species while hexachlorophene at 10(-3) M also caused 100% inhibition in homogenates from Fasciola gagantica and P. explanatum but only 65% of malate oxidation in Fasciolopsis buski homogenates. Nitroxynil at 10(-3) M produced 60% inhibition in F. buski homogenates yet had little effect at this concentration on preparations from the other species. Little inhibition was seen with diamphenethide, even at high concentrations. Rapid death of Fasicola gigantica and P. explanatum resulted in vitro when 10(-3) M oxyclozanide, hexachlorophene, nitroxynil or rafoxanide, were added to the incubation medium. Fasciolopsis buski was killed by 10(-3) M oxyclozanide but at this concentration the remaining compounds only caused reduced activity. Assay of malate dehydrogenase following drug treatment in vitro failed to show any appreciable reduction in enzyme activity in Fasciola gigantica and P. explanatum but oxyclozanide and hexachlorophene produced inhibition in Fasciolopsis buski. The mode of action of these compounds is discussed. PMID:7264272

  13. 17β-Estradiol treatment inhibits breast cell proliferation, migration and invasion by decreasing MALAT-1 RNA level

    SciTech Connect

    Zhao, Ziyi; Chen, Changjin; Liu, Yu; Wu, Chuanfang

    2014-03-07

    Highlights: • E2 affects not only estrogen-receptor α positive breast cells but also negative ones. • 100 nM E2 treatment affects breast cells proliferation, migration. • 100 nM E2 treatment functions in an estrogen-receptor α-independent way. • E2 treatment decreases MALAT-1 RNA level by post-transcriptional regulation. - Abstract: Breast cancer cells, which express estrogen receptor α (ERα), respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. But breast cancer cells without ERα show no effect on low concentration of estrogen treatment. Proliferation, migration and invasion of MCF10a, MCF7 and MB231 cells treated with low (1 nM) or high (100 nM) dose of 17β-Estradiol (E2) was performed. We identified the effects of E2 on these breast cell lines, and looked for the difference in the presence and absence of ERα. Specifically, we looked for the changes of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT-1), which is found extensively and highly expressed in several kinds of tumor cells, including breast carcinoma. It was observed that proliferation, migration and invasion of breast cells were greatly affected by high concentration E2 treatment and were not affected by low concentration E2 treatment in an ERα independent way. We found that the high concentration E2 treatment largely decreased MALAT-1 RNA level. Interestingly, MALAT-1 decreasing by knocking down showed similar effects on proliferation, migration and invasion. E2 treatment affects breast tumor or non-tumor cells proliferation, migration and invasion in an ERα -independent, but a dose-dependent way by decreasing the MALAT-1 RNA level.

  14. Redirection of the phenylpropanoid pathway to feruloyl malate in Arabidopsis mutants deficient for cinnamoyl-CoA reductase 1.

    PubMed

    Mir Derikvand, Mohammad; Sierra, Jimmy Berrio; Ruel, Katia; Pollet, Brigitte; Do, Cao-Trung; Thévenin, Johanne; Buffard, Dominique; Jouanin, Lise; Lapierre, Catherine

    2008-04-01

    Cinnamoyl-CoA reductase 1 (CCR1, gene At1g15950) is the main CCR isoform implied in the constitutive lignification of Arabidopsis thaliana. In this work, we have identified and characterized two new knockout mutants for CCR1. Both have a dwarf phenotype and a delayed senescence. At complete maturity, their inflorescence stems display a 25-35% decreased lignin level, some alterations in lignin structure with a higher frequency of resistant interunit bonds and a higher content in cell wall-bound ferulic esters. Ferulic acid-coniferyl alcohol ether dimers were found for the first time in dicot cell walls and in similar levels in wild-type and mutant plants. The expression of CCR2, a CCR gene usually involved in plant defense, was increased in the mutants and could account for the biosynthesis of lignins in the CCR1-knockout plants. Mutant plantlets have three to four-times less sinapoyl malate (SM) than controls and accumulate some feruloyl malate. The same compositional changes occurred in the rosette leaves of greenhouse-grown plants. By contrast and relative to the control, their stems accumulated unusually high levels of both SM and feruloyl malate as well as more kaempferol glycosides. These findings suggest that, in their hypolignified stems, the mutant plants would avoid the feruloyl-CoA accumulation by its redirection to cell wall-bound ferulate esters, to feruloyl malate and to SM. The formation of feruloyl malate to an extent far exceeding the levels reported so far indicates that ferulic acid is a potential substrate for the enzymes involved in SM biosynthesis and emphasizes the remarkable plasticity of Arabidopsis phenylpropanoid metabolism. PMID:18046574

  15. Thin-film a-Si:H solar cells processed on aluminum-induced texture (AIT) glass superstrates: prediction of light absorption enhancement.

    PubMed

    Sahraei, Nasim; Peters, Marius; Venkataraj, Selvaraj; Aberle, Armin G; Calnan, Sonya; Ring, Sven; Stannowski, Bernd; Schlatmann, Rutger; Stangl, Rolf

    2015-05-10

    Light scattering superstrates are important for thin-film a-Si:H solar cells. In this work, aluminum-induced texture (AIT) glass, covered with nonetched Al-doped ZnO (AZO), is investigated as an alternative to the commonly used planar glass with texture-etched AZO superstrate. Four different AIT glasses with different surface roughnesses and different lateral feature sizes are investigated for their effects on light trapping in a-Si:H solar cells. For comparison, two reference superstrates are investigated as well: planar glass covered with nonetched AZO and planar glass covered with texture-etched AZO. Single-junction a-Si:H solar cells are deposited onto each superstrate, and the scattering properties (haze and angular resolved scattering) as well as the solar cell characteristics (current-voltage and external quantum efficiency) are measured and compared. The results indicate that AIT glass superstrates with nonetched AZO provide similar, or even superior, light trapping than the standard reference superstrate, which is demonstrated by a higher short-circuit current Jsc and a higher external quantum efficiency. Using the trapped light fraction δ, a quantity based on the integrated light scattering at the AZO/a-Si:H interface, we show that Jsc linearly increases with δ in the scattering regime of the samples, regardless of the type of superstrate used. PMID:25967490

  16. Mitoprotective effect of Centella asiatica against aluminum-induced neurotoxicity in rats: possible relevance to its anti-oxidant and anti-apoptosis mechanism.

    PubMed

    Prakash, Atish; Kumar, Anil

    2013-08-01

    Role of mitochondrial dysfunction and oxidative stress has been well documented in various cognitive-related disorders such as Alzheimer's disease. Aluminum is a neurotoxic metal that may be involved in the progression of neurodegenerative processes. The antioxidant and memory enhancing effects of Centella asiatica (CA) are well known in the last few decades. Therefore, the present study has been designed to explore the neuroprotective effect of CA on chronic aluminum exposure induced mitochondrial enzyme alteration, oxidative stress, apoptosis and cognitive dysfunction in rat. Aluminum (100 mg/kg) and CA (150 and 300 mg/kg) were administered daily for a period of 6 weeks in male Wistar rats. Various behavioral, biochemical and cellular estimations and aluminum concentration were assessed. Chronic aluminum administration resulted in memory impairment and caused marked oxidative damage associated with mitochondria impairment. It also caused a significant increase in caspase-3 activity, acetylcholine esterase activity and aluminum concentration in hippocampus and cerebral cortex of rat brain. Chronic administration of CA significantly improved memory performance, oxidative defense decreased aluminum concentration, caspase-3, acetylcholinestrease activity and reversal of mitochondrial enzyme activity as compared to aluminum-treated animals. Results of the study demonstrate neuroprotective potential of CA against aluminum-induced cognitive dysfunction and mito- oxidative damage. PMID:23224641

  17. The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult

    PubMed Central

    Zhang, Bin; Arun, Gayatri; Mao, Yuntao S.; Lazar, Zsolt; Hung, Gene; Bhattacharjee, Gourab; Xiao, Xiaokun; Booth, Carmen J.; Wu, Jie; Zhang, Chaolin; Spector, David L.

    2012-01-01

    SUMMARY Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs) lacking protein coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1) is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3′-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates Malat1 is not essential for mouse pre- and post-natal development. Furthermore, depletion of Malat1 does not impact global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis regulatory role of Malat1 gene transcription. PMID:22840402

  18. 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. PMID:22163924

  19. MALAT1 long ncRNA promotes gastric cancer metastasis by suppressing PCDH10

    PubMed Central

    Qi, Ying; Ooi, Hong Sain; Wu, Jun; Chen, Jian; Zhang, Xiaoli; Tan, Sheng; Yu, Qing; Li, Yuan-Yuan; Kang, Yani; Li, Hua; Xiong, Zirui; Zhu, Tao; Liu, Bingya; Shao, Zhifeng; Zhao, Xiaodong

    2016-01-01

    EZH2, the catalytic component of polycomb repressive complex 2 (PRC2), is frequently overexpressed in human cancers and contributes to tumor initiation and progression, in part through transcriptional silencing of tumor suppressor genes. A number of noncoding RNAs (ncRNAs) recruit EZH2 to specific chromatin loci, where they modulate gene expression. Here, we used RNA immunoprecipitation sequencing (RIP-seq) to profile EZH2-associated transcripts in human gastric cancer cell lines. We identified 8,256 transcripts, including both noncoding and coding transcripts, some of which were derived from cancer-related loci. In particular, we found that long noncoding RNA (lncRNA) MALAT1 binds EZH2, suppresses the tumor suppressor PCDH10, and promotes gastric cellular migration and invasion. Our work thus provides a global view of the EZH2-associated transcriptome and offers new insight into the function of EZH2 in gastric tumorigenesis. PMID:26871474

  20. Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

    SciTech Connect

    Decottignies, P.; Schmitter, J.M.; Miginiac-Maslow, M.; Le Marechal, P.; Jacquot, J.P.; Gadal, P.

    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, the 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.

  1. Hoogsteen-position pyrimidines promote the stability and function of the MALAT1 RNA triple helix.

    PubMed

    Brown, Jessica A; Kinzig, Charles G; DeGregorio, Suzanne J; Steitz, Joan A

    2016-05-01

    Triple-stranded RNA was first deduced to form in vitro more than 50 years ago and has since been implicated in RNA catalysis, stability, and small molecule binding. Despite the emerging biological significance of RNA triple helices, it remains unclear how their nucleotide composition contributes to their thermodynamic stability and cellular function. To investigate these properties, we used in vitro RNA electrophoretic mobility shift assays (EMSAs) and in vivo intronless β-globin reporter assays to measure the relative contribution of 20 RNA base triples (N•A-U, N•G-C, N•C-G, N•U-A, and N•G-U) to triple-helical stability. These triples replaced a single internal U•A-U within the known structure of the triple-helical RNA stability element of human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which contains 10 major-groove base triples. In addition to the canonical C•G-C triple, the noncanonical base triples U•G-C, U•G-U, C•C-G, and U•C-G exhibited at least 30% stability relative to the wild-type U•A-U base triple in both assays. Of these triples, only U•A-U, C•G-C, and U•G-C, when tested as four successive triples, formed stabilizing structures that allowed accumulation of the intronless β-globin reporter. Overall, we find that Hoogsteen-position pyrimidines support triple helix stability and function and that thermodynamic stability, based on EMSA results, is necessary but not sufficient for stabilization activity of the MALAT1 triple helix in cells. These results suggest that additional RNA triple helices containing noncanonical triples likely exist in nature. PMID:26952103

  2. Acute Citrulline-Malate Supplementation and High-Intensity Cycling Performance.

    PubMed

    Cunniffe, Brian; Papageorgiou, Maria; OʼBrien, Barbara; Davies, Nathan A; Grimble, George K; Cardinale, Marco

    2016-09-01

    Cunniffe, B, Papageorgiou, M, O'Brien, B, Davies, NA, Grimble, GK, and Cardinale, M. Acute citrulline-malate supplementation and high-intensity cycling performance. J Strength Cond Res 30(9): 2638-2647, 2016-Dietary L-citrulline-malate (CM) consumption has been suggested to improve skeletal muscle metabolism and contractile efficiency, which would be expected to predispose exercising individuals to greater fatigue resistance. The purpose of this study was to examine the effects of CM supplementation on acid-base balance and high-intensity exercise performance. In a double-blind, placebo-controlled, crossover study, 10 well-trained males consumed either 12 g of CM (in 400 ml) or lemon sugar-free cordial (placebo [PL]) 60 minutes before completion of 2 exercise trials. Each trial consisted of subjects performing 10 (×15 seconds) maximal cycle sprints (with 30-second rest intervals) followed by 5 minutes recovery before completing a cycle time-to-exhaustion test (TTE) at 100% of individual peak power (PP). Significant increases in plasma concentrations of citrulline (8.8-fold), ornithine (3.9-fold), and glutamine (1.3-fold) were observed 60 minutes after supplementation in the CM trial only (p ≤ 0.05) and none of the subjects experienced gastrointestinal side-effects during testing. Significantly higher exercise heart rates were observed in CM condition (vs. PL) although no between trial differences in performance related variables (TTE: [120 ± 61 seconds CM vs. 113 ± 50 seconds PL]), PP or mean power, ([power fatigue index: 36 ± 16% CM vs. 28 ± 18% PL]), subjective rating of perceived exertion or measures of acid-base balance (pH, lactate, bicarbonate, base-excess) were observed (p > 0.05). This study demonstrated that acute supplementation of 12 g CM does not provide acute ergogenic benefits using the protocol implemented in this study in well-trained males. PMID:26808848

  3. Evaluation of the Reproductive Toxicity, Glycometabolism, Glycometabolism-Related Enzyme Levels and Lipid Metabolism of Chromium Malate Supplementation in Sprague-Dawley Rats.

    PubMed

    Feng, Weiwei; Zhang, Weijie; Zhao, Ting; Mao, Guanghua; Wang, Wei; Wu, Xueshan; Zhou, Zhaoxiang; Huang, Jing; Bao, Yongtuan; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the reproductive toxicity of chromium malate in Sprague-Dawley rats and then inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, and lipid metabolism. The results showed that no pathological, toxic feces and urine changes were observed in clinical signs of parental and fetal rats in chromium malate groups. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of chromium malate groups have no significant change compared with control group and chromium picolinate group. The serum and organ contents of Cr in chromium malate groups have no significant change when compared with control group. No measurable damage on liver, brain, kidney, and testis/uterus of chromium malate groups was found. No significant change in body mass, absolute and relative organ weights, and hematological and biochemical changes of rats were observed compared with the control and chromium picolinate groups. The results indicated that supplements with chromium malate does not cause obvious damage and has no obvious effect on glycometabolism, glycometabolism-related enzyme, and lipid metabolism on female and male rats. The results of this study suggested that chromium malate is safe for human consumption and has the potential for application as a functional food ingredient and dietary supplement. PMID:25876088

  4. Nanomolar aluminum induces expression of the inflammatory systemic biomarker C-reactive protein (CRP) in human brain microvessel endothelial cells (hBMECs).

    PubMed

    Alexandrov, Peter N; Kruck, Theodore P A; Lukiw, Walter J

    2015-11-01

    C-reactive protein (CRP; also known as pentraxin 1, PTX1), a 224 amino acid soluble serum protein organized into a novel pentameric ring-shaped structure, is a highly sensitive pathogenic biomarker for systemic inflammation. High CRP levels are found in practically every known inflammatory state, and elevated CRP levels indicate an increased risk for several common age-related human degenerative disorders, including cardiovascular disease, cancer, diabetes, and Alzheimer's disease (AD). While the majority of CRP is synthesized in the liver for secretion into the systemic circulation, it has recently been discovered that an appreciable amount of CRP is synthesized in highly specialized endothelial cells that line the vasculature of the brain and central nervous system (CNS). These highly specialized cells, the major cell type lining the human CNS vasculature, are known as human brain microvessel endothelial cells (hBMECs). In the current pilot study we examined (i) CRP levels in human serum obtained from AD and age-matched control patients; and (ii) analyzed the effects of nanomolar aluminum sulfate on CRP expression in primary hBMECs. The three major findings in this short communication are: (i) that CRP is up-regulated in AD serum; (ii) that CRP serum levels increased in parallel with AD progression; and (iii) for the first time show that nanomolar aluminum potently up-regulates CRP expression in hBMECs to many times its 'basal abundance'. The results suggest that aluminum-induced CRP may in part contribute to a pathophysiological state associated with a chronic systemic inflammation of the human vasculature. PMID:26265215

  5. 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

  6. Engineering a domain-locking disulfide into a bacterial malate dehydrogenase produces a redox-sensitive enzyme.

    SciTech Connect

    Muslin, E. H.; Li, D.; Stevens, F. J.; Donnelly, M.; Schiffer, M.; Anderson, L. E.; Univ. of Illinois at Chicago

    1995-06-01

    Light-dependent reduction of cystine disulfide bonds results in activation of several of the enzymes of photosynthetic carbon metabolism within the chloroplast. We have modeled the tertiary structure of four of these light-activated enzymes, namely NADP-linked malate dehydrogenase, glyceraldehyde-3-P dehydrogenase, fructosebisphosphatase, and sedoheptulosebisphosphatase, and identified cysteines in each enzyme that be expected to form inactivating disulfide bonds (Li, D., F. J. Stevens, M. Schiffer, and L. E. Anderson, 1994. Biophys. J. 67:29-35). We have now converted two residues in the Escherichia coli NAD-linked malate dehydrogenase to cysteines and produced a redox-sensitive enzyme. Oxidation of domain-locking cysteine residues in the mutant enzyme clearly mimics dark inactivation of the redox-sensitive chloroplast dehydrogenase. This result is completely consistent with our proposed mechanism.

  7. NADP-Malate Dehydrogenase Gene Evolution in Andropogoneae (Poaceae): Gene Duplication Followed by Sub-functionalization

    PubMed Central

    RONDEAU, P.; ROUCH, C.; BESNARD, G.

    2005-01-01

    • Background and Aims Plastid NADP-dependent malate dehydrogenase (MDH) catalyses the conversion of oxaloacetate to malate. In C4 plants, it is involved in photosynthetic carbon assimilation. In Poaceae, one NADP-MDH gene has been identified in rice (C3; Erhartoideae) and maize (C4; Panicoideae), whereas two tandemly repeated genes have been identified in Sorghum (C4; Panicoideae). In the present study, the molecular evolution of the NADP-MDH multigene family was investigated in order to analyse how the C4 isoform has evolved over a broader range of panicoid grasses. • Methods Polymerase chain reaction (PCR)-based cloning was used to isolate cDNAs encoding NADP-MDHs from 15 species of Panicoideae. A gene phylogeny was reconstructed based on cDNA sequences using distance and maximum parsimony methods. Episodic selection along some branches of the phylogenetic tree was tested by analysing non-synonymous and synonymous rate ratios.Transcription of NADP-MDH genes was compared in green leaves of five accessions of Saccharum, Sorghum and Vetiveria using a semi-quantitative PCR approach. • Key Results Phylogenetic analyses of these data support the existence of two NADP-MDH gene lineages (NMDH-I and NMDH-II) in several Andropogoneae (i.e. Saccharum, Sorghum and Vetiveria). Episodic positive selection was shown along the basal branch of the NMDH-II clade. Three amino acid modifications allow the two gene lineages to be distinguished, suggesting a positive selection at these sites. In green leaves, we showed that the transcript accumulation was higher for NMDH-I than for NMDH-II. • Conclusions It is hypothesized that the maintenance of both NADP-MDH genes in some Andropogoneae is due to a partition of the original functions across both copies. NMDH-I probably corresponds to the C4 isoform as previously suggested. Nevertheless, some C4 species (e.g. maize) only have one gene which should be selected for its high expression level in leaves. This study confirms that

  8. Type 2 Diabetic Rats on Diet Supplemented With Chromium Malate Show Improved Glycometabolism, Glycometabolism-Related Enzyme Levels and Lipid Metabolism

    PubMed Central

    Feng, Weiwei; Zhao, Ting; Mao, Guanghua; Wang, Wei; Feng, Yun; Li, Fang; Zheng, Daheng; Wu, Huiyu; Jin, Dun; Yang, Liuqing; Wu, Xiangyang

    2015-01-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the effect of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism in type 2 diabetic rats. Our results showed that fasting blood glucose, serum insulin level, insulin resistance index and C-peptide level in the high dose group had a significant downward trend when compared with the model group, chromium picolinate group and chromium trichloride group. The hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, Glut4, phosphor-AMPKβ1 and Akt levels in the high dose group were significantly higher than those of the model, chromium picolinate and chromium trichloride groups. Chromium malate in a high dose group can significantly increase high density lipoprotein cholesterol level while decreasing the total cholesterol, low density lipoprotein cholesterol and triglyceride levels when compared with chromium picolinate and chromium trichloride. The serum chromium content in chromium malate and chromium picolinate group is significantly higher than that of the chromium trichloride group. The results indicated that the curative effects of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism changes are better than those of chromium picolinate and chromium trichloride. Chromium malate contributes to glucose uptake and transport in order to improved glycometabolism and glycometabolism-related enzymes. PMID:25942313

  9. 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

  10. Bacillus subtilis YxkJ is a secondary transporter of the 2-hydroxycarboxylate transporter family that transports L-malate and citrate.

    PubMed

    Krom, B P; Aardema, R; Lolkema, J S

    2001-10-01

    The genome of Bacillus subtilis contains two genes that code for membrane proteins that belong to the 2-hydroxycarboxylate transporter family. Here we report the functional characterization of one of the two, yxkJ, which codes for a transporter protein named CimHbs. The gene was cloned and expressed in Escherichia coli and complemented the citrate-negative phenotype of wild-type E. coli and the malate-negative phenotype of the E. coli strain JRG4008, which is defective in malate uptake. Subsequent uptake studies in whole cells expressing CimHbs clearly demonstrated the citrate and malate transport activity of the protein. Immunoblot analysis showed that CimHbs is a 48-kDa protein that is well expressed in E. coli. Studies with right-side-out membrane vesicles demonstrated that CimHbs is an electroneutral proton-solute symporter. No indications were found for the involvement of Na(+) ions in the transport process. Inhibition of the uptake catalyzed by CimHbs by divalent metal ions, together with the lack of effect on transport by the chelator EDTA, showed that CimHbs translocates the free citrate and malate anions. Among a large set of substrates tested, only malate, citramalate, and citrate competitively inhibited citrate transport catalyzed by CimHbs. The transporter is strictly stereoselective, recognizing only the S enantiomers of malate and citramalate. Remarkably, though citramalate binds to the transporter, it is not translocated. PMID:11566984

  11. Decarboxylation of Malate in the Crassulacean Acid Metabolism Plant Bryophyllum (Kalanchoe) fedtschenkoi (Role of NAD-Malic Enzyme).

    PubMed

    Cook, R. M.; Lindsay, J. G.; Wilkins, M. B.; Nimmo, H. G.

    1995-12-01

    The role of NAD-malic enzyme (NAD-ME) in the Crassulacean acid metabolism plant Bryophyllum (Kalanchoe) fedtschenkoi was investigated using preparations of intact and solubilized mitochondria from fully expanded leaves. Intact, coupled mitochondria isolated during the day or night did not differ in their ability to take up [14C]malic acid from the surrounding medium or to respire using malate or succinate as substrate. However, intact mitochondria isolated from plants during the day decarboxylated added malate to pyruvate significantly faster than mitochondria isolated from plants at night. NAD-ME activity in solubilized mitochondrial extracts showed hysteretic kinetics and was stimulated by a number of activators, including acetyl-coenzyme A, fructose-1,6-bisphosphate, and sulfate ions. In the absence of these effectors, reaction progress curves were nonlinear, with a pronounced acceleration phase. The lag period before a steady-state rate was reached in assays of mitochondrial extracts decreased during the photoperiod and increased slowly during the period of darkness. However, these changes in the kinetic properties of the enzyme could not account for the changes in the rate of decarboxylation of malate by intact mitochondria. Gel-filtration experiments showed that mitochondrial extracts contained three forms of NAD-ME with different molecular weights. The relative proportions of the three forms varied somewhat throughout the light/dark cycle, but this did not account for the changes in the kinetics behavior of the enzyme during the diurnal cycle. PMID:12228671

  12. 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. PMID:23603074

  13. RBM24 suppresses cancer progression by upregulating miR-25 to target MALAT1 in nasopharyngeal carcinoma.

    PubMed

    Hua, Wen-Feng; Zhong, Qian; Xia, Tian-Liang; Chen, Qi; Zhang, Mei-Yin; Zhou, Ai-Jun; Tu, Zi-Wei; Qu, Chen; Li, Man-Zhi; Xia, Yun-Fei; Wang, Hui-Yun; Xie, Dan; Claret, Francois-Xavier; Song, Er-Wei; Zeng, Mu-Sheng

    2016-01-01

    Abnormal interaction between non-coding RNAs has been demonstrated to be a common molecular event in various human cancers, but its significance and underlying mechanisms have not been well documented. RNA-binding proteins (RBPs) are key regulators of RNA transcription and post-transcriptional processing. In this study, we found that RNA-binding protein 24 (RBM24) was frequently downregulated in nasopharyngeal carcinoma (NPC). The restoration of RBM24 expression suppressed NPC cellular proliferation, migration and invasion and impeded metastatic colonization in mouse models. Microarray analyses revealed that miR-25 expression was upregulated by RBM24 expression in NPC cells. Similarly, ectopic miR-25 expression suppressed NPC cellular growth and motility by targeting the pro-oncogenic lncRNA MALAT1, and the knockdown of MALAT1 expression exhibited similar effects as RBM24 restoration in NPC cells. Overall, these findings suggest a novel role of RBM24 as a tumor suppressor. Mechanistically, RBM24 acts at least in part through upregulating the expression of miR-25, which in turn targets MALAT1 for degradation. PMID:27584791

  14. Ab Initio Calculation of the Zn Isotope Effect in Phosphates, Citrates, and Malates and Applications to Plants and Soil

    PubMed Central

    Fujii, Toshiyuki; Albarède, Francis

    2012-01-01

    Stable Zn isotopes are fractionated in roots and leaves of plants. Analyses demonstrate that the heavy Zn isotopes are enriched in the root system of plants with respect to shoots and leaves as well as the host soil, but the fractionation mechanisms remain unclear. Here we show that the origin of this isotope fractionation is due to a chemical isotope effect upon complexation by Zn malates and citrates in the aerial parts and by phosphates in the roots. We calculated the Zn isotope effect in aqueous citrates, malates, and phosphates by ab initio methods. For pH<5, the Zn isotopic compositions of the various parts of the plants are expected to be similar to those of groundwater. In the neutral to alkaline region, the calculations correctly predict that 66Zn is enriched over 64Zn in roots, which concentrate phosphates, with respect to leaves, which concentrate malates and citrates, by about one permil. It is proposed that Zn isotope fractionation represents a useful tracer of Zn availability and mobility in soils. PMID:22363478

  15. Decarboxylation of Malate in the Crassulacean Acid Metabolism Plant Bryophyllum (Kalanchoe) fedtschenkoi (Role of NAD-Malic Enzyme).

    PubMed Central

    Cook, R. M.; Lindsay, J. G.; Wilkins, M. B.; Nimmo, H. G.

    1995-01-01

    The role of NAD-malic enzyme (NAD-ME) in the Crassulacean acid metabolism plant Bryophyllum (Kalanchoe) fedtschenkoi was investigated using preparations of intact and solubilized mitochondria from fully expanded leaves. Intact, coupled mitochondria isolated during the day or night did not differ in their ability to take up [14C]malic acid from the surrounding medium or to respire using malate or succinate as substrate. However, intact mitochondria isolated from plants during the day decarboxylated added malate to pyruvate significantly faster than mitochondria isolated from plants at night. NAD-ME activity in solubilized mitochondrial extracts showed hysteretic kinetics and was stimulated by a number of activators, including acetyl-coenzyme A, fructose-1,6-bisphosphate, and sulfate ions. In the absence of these effectors, reaction progress curves were nonlinear, with a pronounced acceleration phase. The lag period before a steady-state rate was reached in assays of mitochondrial extracts decreased during the photoperiod and increased slowly during the period of darkness. However, these changes in the kinetic properties of the enzyme could not account for the changes in the rate of decarboxylation of malate by intact mitochondria. Gel-filtration experiments showed that mitochondrial extracts contained three forms of NAD-ME with different molecular weights. The relative proportions of the three forms varied somewhat throughout the light/dark cycle, but this did not account for the changes in the kinetics behavior of the enzyme during the diurnal cycle. PMID:12228671

  16. Staphylococcus aureus lactate- and malate-quinone oxidoreductases contribute to nitric oxide resistance and virulence.

    PubMed

    Spahich, Nicole A; Vitko, Nicholas P; Thurlow, Lance R; Temple, Brenda; Richardson, Anthony R

    2016-06-01

    Staphylococcus aureus is a Gram-positive pathogen that resists many facets of innate immunity including nitric oxide (NO·). Staphylococcus aureus NO-resistance stems from its ability to evoke a metabolic state that circumvents the negative effects of reactive nitrogen species. The combination of l-lactate and peptides promotes S. aureus growth at moderate NO-levels, however, neither nutrient alone suffices. Here, we investigate the staphylococcal malate-quinone and l-lactate-quinone oxidoreductases (Mqo and Lqo), both of which are critical during NO-stress for the combined utilization of peptides and l-lactate. We address the specific contributions of Lqo-mediated l-lactate utilization and Mqo-dependent amino acid consumption during NO-stress. We show that Lqo conversion of l-lactate to pyruvate is required for the formation of ATP, an essential energy source for peptide utilization. Thus, both Lqo and Mqo are essential for growth under these conditions making them attractive candidates for targeted therapeutics. Accordingly, we exploited a modelled Mqo/Lqo structure to define the catalytic and substrate-binding residues.We also compare the S. aureus Mqo/Lqo enzymes to their close relatives throughout the staphylococci and explore the substrate specificities of each enzyme. This study provides the initial characterization of the mechanism of action and the immunometabolic roles for a newly defined staphylococcal enzyme family. PMID:26851155

  17. Purification and characterization of the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana.

    PubMed

    An, Yan; Cao, Youzhi; Xu, Yingwu

    2016-07-01

    Malate dehydrogenase (MDH) ubiquitously exists in living organisms and has many isoforms in a single species. MDHs from some classes have been characterized for their catalytic properties, which show significant variations despite that they share high sequence identity for the active sites. One class MDH, the plastid-localized NAD-dependent MDH (plNAD-MDH) is known to be important for plant survival in a dark environment, but its biochemical and enzymatic properties have not been well characterized. This study attempts to fill the gap. plNAD-MDH was expressed in an Escherichia coli system and purified using nickel-affinity chromatography followed by size exclusion chromatography. The N-terminal fusion his-tag was removed by protease cleavage. The gel filtration assay and glutaraldehyde cross-linking results showed that the active enzyme was a homodimer in solution. Further assay indicated that plNAD-MDH is most active at a neutral pH value. The Km values for oxaloacetate and NADH are found in the submillimolar order, a median range for most MDHs. The maximum reaction rate values, however, are dramatically different from other plant MDHs, indicating that plNAD-MDH has different substrate specificity. Moreover, we obtained crystals for this enzyme, which laid the groundwork for further analysis of the enzymatic mechanism from structural stand point. PMID:26095832

  18. Synthesis and crystal structures of two nickel coordination polymers generated from asymmetric malate ligand

    SciTech Connect

    Guo Yaqin; Xiao Dongrong; Wang Enbo . E-mail: wangenbo@public.cc.jl.cn; Lu Ying; Lue Jian; Xu Xinxin; Xu Lin

    2005-03-15

    Two nickel coordination polymers [Ni(H{sub 2}O)(C{sub 4}H{sub 4}O{sub 5})].H{sub 2}O 1 and [Ni(H{sub 2}O)(mal)(phen)] 2, have been hydrothermally synthesized and structurally characterized by single crystal X-ray diffraction. Crystal data for 1: C{sub 4}H{sub 8}O{sub 7}Ni, monoclinic Cc, a=13.156(3)A, b=7.5436(15)A, c=9.6982(19)A, {beta}=130.96(3){sup o}, Z=4. Crystal data for 2: C{sub 16}H{sub 14}N{sub 2}O{sub 6}Ni, orthorhombic Pna2{sub 1}, a=9.6113(19)A, b=19.691(4)A, c=8.0944(16)A, Z=4. Compound 1 is constructed from [Ni(H{sub 2}O)(C{sub 4}H{sub 4}O{sub 5})] sheets pillared through {beta}-carboxylate groups into a 3D framework, which exhibits a diamond-like network. Compound 2 exhibits a 3D supramolecular network. To our knowledge, compound 1 represents the first diamond-like topology in the system of metal-malate. Other characterizations by elemental analysis, IR and TG are also described. The magnetic behavior of compound 1 has been studied.

  19. [Implication of glutamate, isocitrate and malate deshydrogenases in nitrogen assimilation in the cadmium-stressed tomato].

    PubMed

    Chaffei, Chiraz; Suzuki, Akira; Masclaux-Daubresse, Céline; Ghorbel, Mohamed Habib; Gouia, Houda

    2006-10-01

    Tomato seedlings grown on nitric medium and treated with various cadmium concentrations (0 to 50 microM) were used. Results obtained show that cadmium remains predominantly located in the roots, which then seem to play the role of trap-organs. Increasing cadmium concentration in the medium leads particularly to a decrease in NO3- accumulation, together with a decrease in the activity of glutamine synthetase and in the quantity of plastidic isoform ARNm (GS2), and, on the contrary, to an increase of the cytosolic isoform ARNm (GS1). On the other hand, stimulations were observed for NADH-dependent glutamate synthase, NADH-dependent glutamate dehydrogenase, ARNm quantity of this enzyme, ammonium accumulation, and protease activity. In parallel, stimulations were observed for NAD+ and NADP+-dependent malate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. These results were discussed in relation to the hypothesis attributing to the dehydrogenase enzymes (GDH, MDH, ICDH) an important role in the plant defence processes against cadmium-induced stresses. PMID:17027640

  20. A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating Mitochondrial Respiration.

    PubMed

    Ban, Hyun Seung; Xu, Xuezhen; Jang, Kusik; Kim, Inhyub; Kim, Bo-Kyung; Lee, Kyeong; Won, Misun

    2016-01-01

    We previously reported that hypoxia-inducible factor (HIF)-1 inhibitor LW6, an aryloxyacetylamino benzoic acid derivative, inhibits malate dehydrogenase 2 (MDH2) activity during the mitochondrial tricarboxylic acid (TCA) cycle. In this study, we present a novel MDH2 inhibitor compound 7 containing benzohydrazide moiety, which was identified through structure-based virtual screening of chemical library. Similar to LW6, compound 7 inhibited MDH2 activity in a competitive fashion, thereby reducing NADH level. Consequently, compound 7 reduced oxygen consumption and ATP production during the mitochondrial respiration cycle, resulting in increased intracellular oxygen concentration. Therefore, compound 7 suppressed the accumulation of HIF-1α and expression of its target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). Moreover, reduction in ATP content activated AMPK, thereby inactivating ACC and mTOR the downstream pathways. As expected, compound 7 exhibited significant growth inhibition of human colorectal cancer HCT116 cells. Compound 7 demonstrated substantial anti-tumor efficacy in an in vivo xenograft assay using HCT116 mouse model. Taken together, a novel MDH2 inhibitor, compound 7, suppressed HIF-1α accumulation via reduction of oxygen consumption and ATP production, integrating metabolism into anti-cancer efficacy in cancer cells. PMID:27611801

  1. Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant

    SciTech Connect

    Tomita, Takeo; Fushinobu, Shinya; Kuzuyama, Tomohisa; Nishiyama, Makoto . E-mail: umanis@mail.ecc.u-tokyo.ac.jp

    2006-08-25

    To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant.

  2. Ectopic Expression of the Chinese Cabbage Malate Dehydrogenase Gene Promotes Growth and Aluminum Resistance in Arabidopsis.

    PubMed

    Li, Qing-Fei; Zhao, Jing; Zhang, Jing; Dai, Zi-Hui; Zhang, Lu-Gang

    2016-01-01

    Malate dehydrogenases (MDHs) are key metabolic enzymes that play important roles in plant growth and development. In the present study, we isolated the full-length and coding sequences of BraMDH from Chinese cabbage [Brassica campestris L. ssp. pekinensis (Lour) Olsson]. We conducted bioinformatics analysis and a subcellular localization assay, which revealed that the BraMDH gene sequence contained no introns and that BraMDH is localized to the chloroplast. In addition, the expression pattern of BraMDH in Chinese cabbage was investigated, which revealed that BraMDH was heavily expressed in inflorescence apical meristems, as well as the effect of BraMDH overexpression in two homozygous transgenic Arabidopsis lines, which resulted in early bolting and taller inflorescence stems. Furthermore, the fresh and dry weights of aerial tissue from the transgenic Arabidopsis plants were significantly higher than those from the corresponding wild-type plants, as were plant height, the number of rosette leaves, and the number of siliques produced, and the transgenic plants also exhibited stronger aluminum resistance when treated with AlCl3. Therefore, our results suggest that BraMDH has a dramatic effect on plant growth and that the gene is involved in both plant growth and aluminum resistance. PMID:27536317

  3. Induction of Malate Synthase Gene Expression in Senescent and Detached Organs of Cucumber.

    PubMed Central

    Graham, IA; Leaver, CJ; Smith, SM

    1992-01-01

    Expression of the malate synthase (MS) gene is activated in cotyledons of cucumber seedlings during postgerminative growth and then repressed as the cotyledons become photosynthetic. MS gene expression is subsequently reactivated in the cotyledons as they senesce a few weeks later. In situ hybridization revealed that MS RNA is distributed throughout the organ during postgerminative growth and senescence, showing that the same cells express the gene at different stages of development. MS RNA also appears in senescing leaves and petals of cucumber plants. In addition, we found that MS RNA appears in mature expanded leaves and roots when they are removed from the plant and incubated in darkness for several days, thus providing a potential experimental system for the manipulation of MS gene expression. Leaves from transgenic Nicotiana plumbaginifolia containing the cucumber MS promoter fused to the [beta]-glucuronidase (GUS) reporter gene accumulated GUS activity when detached, demonstrating an activation of transcription from the MS promoter following leaf excision. These results are discussed in terms of the metabolic regulation of MS gene expression. PMID:12297649

  4. High Performance Liquid Chromatographic Analysis of Almotriptan Malate in Bulk and Tablets

    PubMed Central

    Lavudu, Petikam; Rani, Avula Prameela; Divya, Chepuri; Sekharan, Chandra Bala

    2013-01-01

    Purpose: A simple RP-HPLC method has been developed and validated for the determination of almotriptan malate (ATM) in bulk and tablets. Methods: Chromatographic separation of ATM was achieved by using a Thermo Scientific C18 column. A Mobile phase containing a mixture of methanol, water and acetic acid (4:8:0.1 v/v) was pumped at the flow rate of 1 mL/min. Detection was performed at 227 nm. According to ICH guidelines, the method was validated. Results: The calibration curve was linear in the concentration range 5–60 µg/mL for the ATM with regression coefficient 0.9999. The method was precise with RSD <1.2%. Excellent recoveries of 99.60 - 100.80% proved the accuracy of the method. The limits of detection and quantification were found to be 0.025 and 0.075 µg/mL, respectively. Conclusion: The method was successfully applied for the quantification of ATM in tablets with acceptable accuracy and precision. PMID:24312833

  5. Disruption of malate:quinone oxidoreductase increases L-lysine production by Corynebacterium glutamicum.

    PubMed

    Mitsuhashi, Satoshi; Hayashi, Mikiro; Ohnishi, Junko; Ikeda, Masato

    2006-11-01

    Genomic analysis of a classically derived L-lysine-producing mutant, Corynebacterium glutamicum B-6, identified a nonsense mutation in the mqo gene, which encodes malate:quinone oxidoreductase (MQO). The effect of mqo disruption on L-lysine production was investigated in a defined L-lysine producer, C. glutamicum AHP-3, showing approximately 18% increased production. To explore the underlying mechanisms of the increase, the mqo-disrupted strain was analyzed from the viewpoints of redox balance, activities of membrane-bound dehydrogenases, and transcriptome. The intracellular [NADH]/[NAD] ratio in the strain remained unchanged. Also, there were no significant differences in the activities of the membrane-bound dehydrogenases examined. However, transcriptome analysis showed that some TCA cycle genes, such as acn, sucC, and sucD, were down-regulated in the strain. These results suggest that the loss of MQO activity down-regulates the flux of the TCA cycle to maintain the redox balance and results in redirection of oxaloacetate into L-lysine biosynthesis. PMID:17090916

  6. Ectopic Expression of the Chinese Cabbage Malate Dehydrogenase Gene Promotes Growth and Aluminum Resistance in Arabidopsis

    PubMed Central

    Li, Qing-Fei; Zhao, Jing; Zhang, Jing; Dai, Zi-Hui; Zhang, Lu-Gang

    2016-01-01

    Malate dehydrogenases (MDHs) are key metabolic enzymes that play important roles in plant growth and development. In the present study, we isolated the full-length and coding sequences of BraMDH from Chinese cabbage [Brassica campestris L. ssp. pekinensis (Lour) Olsson]. We conducted bioinformatics analysis and a subcellular localization assay, which revealed that the BraMDH gene sequence contained no introns and that BraMDH is localized to the chloroplast. In addition, the expression pattern of BraMDH in Chinese cabbage was investigated, which revealed that BraMDH was heavily expressed in inflorescence apical meristems, as well as the effect of BraMDH overexpression in two homozygous transgenic Arabidopsis lines, which resulted in early bolting and taller inflorescence stems. Furthermore, the fresh and dry weights of aerial tissue from the transgenic Arabidopsis plants were significantly higher than those from the corresponding wild-type plants, as were plant height, the number of rosette leaves, and the number of siliques produced, and the transgenic plants also exhibited stronger aluminum resistance when treated with AlCl3. Therefore, our results suggest that BraMDH has a dramatic effect on plant growth and that the gene is involved in both plant growth and aluminum resistance. PMID:27536317

  7. Cloning, overexpression, purification and crystallization of malate dehydrogenase from Thermus thermophilus

    PubMed Central

    Chang, Yu-Yung; Hung, Chih-Hung; Hwang, Tzann-Shun; Hsu, Chun-Hua

    2013-01-01

    Malate dehydrogenase (MDH) has been used as a conjugate for enzyme immunoassay of a wide variety of compounds, such as drugs of abuse, drugs used in repetitive therapeutic application and hormones. In consideration of the various biotechnological applications of MDH, investigations of MDH from Thermus thermophilus were carried out to further understand the properties of this enzyme. The DNA fragment containing the open reading frame of mdh was amplified from the genomic DNA of T. thermophilus and cloned into the expression vector pET21b(+). The protein was expressed in a soluble form in Escherichia coli strain BL21(DE3). Homogeneous protein was obtained using a three-step procedure consisting of thermal treatment, Ni2+-chelating chromatography and size-exclusion chromatography. The purified MDH was crystallized and the crystals diffracted to a resolution of 1.80 Å on the BL13C1 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 71.3, b = 86.1, c = 118.2 Å. The unit-cell volume of the crystal is compatible with the presence of two monomers in the asymmetric unit, with a corresponding Matthews coefficient V M of 2.52 Å3 Da−1 and a solvent content of 51.2%. The crystal structure of MDH has been solved by molecular replacement and is currently under refinement. PMID:24192361

  8. Protein solvent and weak protein protein interactions in halophilic malate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Ebel, Christine; Faou, Pierre; Zaccai, Giuseppe

    1999-01-01

    With the aim to correlate the solvation, stability and solubility properties of halophilic malate dehydrogenase, we characterized its weak interparticle interactions by small-angle neutron scattering in various solvents. The protein concentration dependence of the apparent radius of gyration and forward scattered intensity extrapolated from Guinier plots, and thus the second virial coefficient, A2, were determined for each solvent condition. In NaCl 1M+2-methylpentane-2,4-diol 30%, a solvent that promotes protein crystallization, A2 is negative, -0.4×10 -4 ml mol g -2 and indicating attractive interactions; in ammonium sulfate 3M, in which the protein precipitates at high concentrations, A2˜0. In 2-5M NaCl, 1-3.5M NaOAc, 1-4.5M KF or 1-2M (NH 4) 2SO 4, in which the protein is very soluble, A2 is positive with a value of the order of 0.4×10 -4 ml mol g -2 which decreases with increasing salt concentration. In MgCl 2 however, A2 increases with increasing salt concentration from 0.2 to 1.3M.

  9. Alternative Oxidase Pathway Optimizes Photosynthesis During Osmotic and Temperature Stress by Regulating Cellular ROS, Malate Valve and Antioxidative Systems.

    PubMed

    Dinakar, Challabathula; Vishwakarma, Abhaypratap; Raghavendra, Agepati S; Padmasree, Kollipara

    2016-01-01

    The present study reveals the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under osmotic and temperature stress conditions in the mesophyll protoplasts of Pisum sativum. The responses of photosynthesis and respiration were monitored at saturating light intensity of 1000 μmoles m(-2) s(-1) at 25°C under a range of sorbitol concentrations from 0.4 to 1.0 M to induce hyper-osmotic stress and by varying the temperature of the thermo-jacketed pre-incubation chamber from 25 to 10°C to impose sub-optimal temperature stress. Compared to controls (0.4 M sorbitol and 25°C), the mesophyll protoplasts showed remarkable decrease in NaHCO3-dependent O2 evolution (indicator of photosynthetic carbon assimilation), under both hyper-osmotic (1.0 M sorbitol) and sub-optimal temperature stress conditions (10°C), while the decrease in rates of respiratory O2 uptake were marginal. The capacity of AOX pathway increased significantly in parallel to increase in intracellular pyruvate and reactive oxygen species (ROS) levels under both hyper-osmotic stress and sub-optimal temperature stress under the background of saturating light. The ratio of redox couple (Malate/OAA) related to malate valve increased in contrast to the ratio of redox couple (GSH/GSSG) related to antioxidative system during hyper-osmotic stress. Further, the ratio of GSH/GSSG decreased in the presence of sub-optimal temperature, while the ratio of Malate/OAA showed no visible changes. Also, the redox ratios of pyridine nucleotides increased under hyper-osmotic (NADH/NAD) and sub-optimal temperature (NADPH/NADP) stresses, respectively. However, upon restriction of AOX pathway by using salicylhydroxamic acid (SHAM), the observed changes in NaHCO3-dependent O2 evolution, cellular ROS, redox ratios of Malate/OAA, NAD(P)H/NAD(P) and GSH/GSSG were further aggravated under stress conditions with concomitant modulations in NADP-MDH and antioxidant enzymes. Taken together, the results indicated

  10. Alternative Oxidase Pathway Optimizes Photosynthesis During Osmotic and Temperature Stress by Regulating Cellular ROS, Malate Valve and Antioxidative Systems

    PubMed Central

    Vishwakarma, Abhaypratap; Raghavendra, Agepati S.; Padmasree, Kollipara

    2016-01-01

    The present study reveals the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under osmotic and temperature stress conditions in the mesophyll protoplasts of Pisum sativum. The responses of photosynthesis and respiration were monitored at saturating light intensity of 1000 μmoles m–2 s–1 at 25°C under a range of sorbitol concentrations from 0.4 to 1.0 M to induce hyper-osmotic stress and by varying the temperature of the thermo-jacketed pre-incubation chamber from 25 to 10°C to impose sub-optimal temperature stress. Compared to controls (0.4 M sorbitol and 25°C), the mesophyll protoplasts showed remarkable decrease in NaHCO3-dependent O2 evolution (indicator of photosynthetic carbon assimilation), under both hyper-osmotic (1.0 M sorbitol) and sub-optimal temperature stress conditions (10°C), while the decrease in rates of respiratory O2 uptake were marginal. The capacity of AOX pathway increased significantly in parallel to increase in intracellular pyruvate and reactive oxygen species (ROS) levels under both hyper-osmotic stress and sub-optimal temperature stress under the background of saturating light. The ratio of redox couple (Malate/OAA) related to malate valve increased in contrast to the ratio of redox couple (GSH/GSSG) related to antioxidative system during hyper-osmotic stress. Further, the ratio of GSH/GSSG decreased in the presence of sub-optimal temperature, while the ratio of Malate/OAA showed no visible changes. Also, the redox ratios of pyridine nucleotides increased under hyper-osmotic (NADH/NAD) and sub-optimal temperature (NADPH/NADP) stresses, respectively. However, upon restriction of AOX pathway by using salicylhydroxamic acid (SHAM), the observed changes in NaHCO3-dependent O2 evolution, cellular ROS, redox ratios of Malate/OAA, NAD(P)H/NAD(P) and GSH/GSSG were further aggravated under stress conditions with concomitant modulations in NADP-MDH and antioxidant enzymes. Taken together, the results indicated

  11. Knockdown of long non-coding RNA MALAT1 increases the blood-tumor barrier permeability by up-regulating miR-140.

    PubMed

    Ma, Jun; Wang, Ping; Yao, Yilong; Liu, Yunhui; Li, Zhen; Liu, Xiaobai; Li, Zhiqing; Zhao, Xihe; Xi, Zhuo; Teng, Hao; Liu, Jing; Xue, Yixue

    2016-02-01

    The blood-tumor barrier (BTB) forms a major obstacle in brain tumor therapy by preventing the delivery of sufficient quantities of therapeutic drugs. Long non-coding RNAs (lncRNAs) play important roles in both normal development and diseases including cancer. Here, we elucidated the expression of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) and defined its functional role in the regulation of BTB function as well as its possible molecular mechanisms. Our results proved that MALAT1 expression was up-regulated in brain microvessels of human glioma and glioma endothelial cells (GECs) which were obtained by co-culturing endothelial cells with glioma cells. Functionally, knockdown of MALAT1 resulted in an impairment and increased the permeability of BTB as well as decreased the expression of ZO-1, occludin and claudin-5 in GECs. Further, there was reciprocal repression between MALAT1 and miR-140, and miR-140 mediated the effects that MALAT1 knockdown exerted. Mechanistic investigations defined that nuclear factor YA (NFYA), a CCAAT box-binding transcription factor, was a direct and functional downstream target of miR-140, which was involved in the MALAT1 knockdown induced regulation of BTB function. Furthermore, NFYA could up-regulate the promoter activities and bind to the promoters of ZO-1, occludin and claudin-5 in GECs. Taken together, we have demonstrated the fact that knockdown of MALAT1 resulted in the increased permeability of BTB, which might contribute to establishing potential therapeutic strategies for human gliomas. PMID:26619802

  12. Proton and anion transport at the tonoplast in crassulacean-acid-metabolism plants: specificity of the malate-influx system in Kalanchoë daigremontiana.

    PubMed

    White, P J; Smith, J A

    1989-09-01

    Tonoplast vesicles were prepared from leaf mesophyll homogenates of the crassulacean-acid-metabolism plant Kalanchoë daigremontiana Hamet et Perrier de la Bâthie to study the effects of anions on ATP- and inorganic-pyrophosphate (PPi)-dependent H(+) transport. In the presence of gramicidin, substrate hydrolysis by the tonoplast ATPase was characteristically stimulated by chloride and inhibited by nitrate, but was unaffected by malate and a wide range of other organic-acid anions; the PPiase was anion-insensitive. Malate was more effective than chloride both in stimulating ATP- and PPi-dependent vesicle acidification (measured as quinacrine-fluorescence quenching) and in dissipating a pre-existing inside-positive membrane potential (measured as oxonol-V-fluorescence quenching), indicating that malate was more readily transported across the tonoplast. Certain other four-carbon dicarboxylates also supported high rates of vesicle acidification, their order of effectiveness being fumarate ≫ malate ∼-succinate > oxalacetate ∼- tartrate; the five-carbon dicarboxylates 2-oxoglutarate and glutarate were also transported, although at lower rates. Experiments with non-naturally occurring anions indicated that the malate transporter was not stereospecific, but that it required the trans-carboxyl configuration for transport. Shorter-chain or longer-chain dicarboxylates were not transported, and neither were monocarboxylates, the amino-acid anions aspartate and glutamate, nor the tricarboxylate isocitrate. The non-permeant anions maleate and tartronate appeared to be competitive inhibitors of malate transport but did not affect chloride transport, indicating that malate and chloride influx at the tonoplast might be mediated by separate transporters. PMID:24201527

  13. Gradual neofunctionalization in the convergent evolution of trichomonad lactate and malate dehydrogenases

    PubMed Central

    Steindel, Phillip A.; Chen, Emily H.; Wirth, Jacob D.

    2016-01-01

    Abstract Lactate and malate dehydrogenases (LDH and MDH) are homologous, core metabolic enzymes common to nearly all living organisms. LDHs have evolved convergently from MDHs at least four times, achieving altered substrate specificity by a different mechanism each time. For instance, the LDH of anaerobic trichomonad parasites recently evolved independently from an ancestral trichomonad MDH by gene duplication. LDH plays a central role in trichomonad metabolism by catalyzing the reduction of pyruvate to lactate, thereby regenerating the NAD+ required for glycolysis. Using ancestral reconstruction methods, we identified the biochemical and evolutionary mechanisms responsible for this convergent event. The last common ancestor of these enzymes was a highly specific MDH, similar to modern trichomonad MDHs. In contrast, the LDH lineage evolved promiscuous activity by relaxing specificity in a gradual process of neofunctionalization involving one highly detrimental substitution at the “specificity residue” (R91L) and many additional mutations of small effect. L91 has different functional consequences in LDHs and in MDHs, indicating a prominent role for epistasis. Crystal structures of modern‐day and ancestral enzymes show that the evolution of substrate specificity paralleled structural changes in dimerization and α‐helix orientation. The relatively small “specificity residue” of the trichomonad LDHs can accommodate a range of substrate sizes and may permit solvent to access the active site, both of which promote substrate promiscuity. The trichomonad LDHs present a multi‐faceted counterpoint to the independent evolution of LDHs in other organisms and illustrate the diverse mechanisms by which protein function, structure, and stability coevolve. PMID:26889885

  14. Presence of two transcribed malate synthase genes in an n-alkane-utilizing yeast, Candida tropicalis.

    PubMed

    Hikida, M; Atomi, H; Fukuda, Y; Aoki, A; Hishida, T; Teranishi, Y; Ueda, M; Tanaka, A

    1991-12-01

    The presence of two genomic DNA regions encoding malate synthase (MS) was shown by Southern blot analysis of the genomic DNA from an n-alkane-assimilating yeast, Candida tropicalis, using a partial MS cDNA probe, in accordance with the fact that two types of partial MS cDNAs have previously been isolated. This was also confirmed by the restriction mapping of the two genes screened from the yeast lambda EMBL library. Nucleotide sequence analysis of the respective genomic DNAs, named MS-1 gene and MS-2 gene, revealed that both regions encoding MS had the same length of 1,653 base pairs, corresponding to 551 amino acids (molecular mass of MS-1, 62,448 Da; MS-2, 62,421 Da). Although 29 nucleotide pairs differed in the sequences of the coding regions, the number of amino acid replacements was only one: 159Asn (MS-1)----159Ser (MS-2). In the 5'-flanking regions, there were replacements of four nucleotide pairs, deletion of one pair, and insertion of four pairs. In spite of the fact that two genomic genes were present and transcribed, RNA blot analysis demonstrated that only one band (about 2 kb) was observable even when the carbon sources in the cultivation medium were changed. A comparison of the amino acid sequences was made with MSs of rape (Brassica napus L.), cucumber seed, pumpkin seed, Escherichia coli, and Hansenula polymorpha. A high homology was observed among these enzymes, the results indicating that the protein structure was relatively well conserved through the evolution of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1794980

  15. Characterization of the N-acetyl-α-D-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis .

    PubMed

    Parsonage, Derek; Newton, Gerald L; Holder, Robert C; Wallace, Bret D; Paige, Carleitta; Hamilton, Chris J; Dos Santos, Patricia C; Redinbo, Matthew R; Reid, Sean D; Claiborne, Al

    2010-09-28

    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 α-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 (→GlcNAc-malate) and the BaBshB deacetylase (→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 ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of l-malate, but not other α-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. PMID:20799687

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

    SciTech Connect

    Parsonage, Derek; Newton, Gerald L.; Holder, Robert C.; Wallace, Bret D.; Paige, Carleitta; Hamilton, Chris J.; Dos Santos, Patricia C.; Redinbo, Matthew R.; Reid, Sean D.; Claiborne, Al

    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 ternary 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.

  17. 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

  18. Formation of triple-helical structures by the 3′-end sequences of MALAT1 and MENβ noncoding RNAs

    PubMed Central

    Brown, Jessica A.; Valenstein, Max L.; Yario, Therese A.; Tycowski, Kazimierz T.; Steitz, Joan A.

    2012-01-01

    Stability of the long noncoding-polyadenylated nuclear (PAN) RNA from Kaposi's sarcoma-associated herpesvirus is conferred by an expression and nuclear retention element (ENE). The ENE protects PAN RNA from a rapid deadenylation-dependent decay pathway via formation of a triple helix between the U-rich internal loop of the ENE and the 3′-poly(A) tail. Because viruses borrow molecular mechanisms from their hosts, we searched highly abundant human long-noncoding RNAs and identified putative ENE-like structures in metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) RNAs. Unlike the PAN ENE, the U-rich internal loops of both predicted cellular ENEs are interrupted by G and C nucleotides and reside upstream of genomically encoded A-rich tracts. We confirmed the ability of MALAT1 and MENβ sequences containing the predicted ENE and A-rich tract to increase the levels of an intronless β-globin reporter RNA. UV thermal denaturation profiles at different pH values support formation of a triple-helical structure composed of multiple U•A-U base triples and a single C•G-C base triple. Additional analyses of the MALAT1 ENE revealed that robust stabilization activity requires an intact triple helix, strong stems at the duplex-triplex junctions, a G-C base pair flanking the triplex to mediate potential A-minor interactions, and the 3′-terminal A of the A-rich tract to form a blunt-ended triplex lacking unpaired nucleotides at the duplex-triplex junction. These examples of triple-helical, ENE-like structures in cellular noncoding RNAs, are unique. PMID:23129630

  19. Recurrent MALAT1-GLI1 oncogenic fusion and GLI1 up-regulation define a subset of plexiform fibromyxoma.

    PubMed

    Spans, Lien; Fletcher, Christopher Dm; Antonescu, Cristina R; Rouquette, Alexandre; Coindre, Jean-Michel; Sciot, Raf; Debiec-Rychter, Maria

    2016-07-01

    Plexiform fibromyxomas are rare neoplasms, being officially recognized as a distinct entity among benign mesenchymal gastric tumours in the 2010 WHO Classification of Tumours of the Digestive System. Characteristically, these tumours have a multinodular/plexiform growth pattern, and histologically contain variably cellular areas of bland myofibroblastic-type spindle cells embedded in an abundant myxoid matrix, rich in capillary-type vessels. As yet, the molecular and/or genetic features of these tumours are unknown. Here we describe a recurrent translocation, t(11;12)(q11;q13), involving the long non-coding gene metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and the gene glioma-associated oncogene homologue 1 (GLI1) in a subgroup of these tumours. The presence of the fusion transcript in our index case was confirmed using polymerase chain reaction (PCR) on genomic DNA, followed by Sanger sequencing. We showed that the truncated GLI1 protein is overexpressed and retains its capacity to transcriptionally activate its target genes. A specific FISH assay was developed to detect the novel MALAT1-GLI1 translocation in formalin-fixed, paraffin-embedded (FFPE) material. This resulted in the identification of two additional cases with this fusion and two cases with polysomy of the GLI1 gene. Finally, immunohistochemistry revealed that the GLI1 protein is exclusively overexpressed in those cases that harbour GLI1/12q13 genomic alterations. In conclusion, overexpression of GLI1 through a recurrent MALAT1-GLI1 translocation or GLI1 up-regulation delineates a pathogenically distinct subgroup of plexiform fibromyxomas with activation of the Sonic Hedgehog signalling pathway. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27101025

  20. 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. PMID:26637549

  1. Jerusalem artichoke mitochondria can export reducing equivalents in the form of malate as a result of D-lactate uptake and metabolism.

    PubMed

    de Bari, Lidia; Valenti, Daniela; Pizzuto, Roberto; Paventi, Gianluca; Atlante, Anna; Passarella, Salvatore

    2005-10-01

    We found that as a result of d-lactate uptake and metabolism by Jerusalem artichoke mitochondria, reducing equivalents were exported from the mitochondrial matrix to the exterior in the form of malate. The rate of malate efflux, as measured photometrically using NADP+ and malic enzyme, depended on the rate of transport across the mitochondrial membrane. It showed saturation characteristics (K(m) = 5 mM; V(max) = 9 nmol/min mg of mitochondrial protein) and was inhibited by non-penetrant compounds. We conclude that reducing equivalent export from mitochondria is due to the occurrence of a putative d-lactate/malate antiporter which differs from other mitochondrial carriers, as shown by the different inhibitor sensitivity. PMID:16129093

  2. Plant sunscreens in the UV-B: ultraviolet spectroscopy of jet-cooled sinapoyl malate, sinapic acid, and sinapate ester derivatives.

    PubMed

    Dean, Jacob C; Kusaka, Ryoji; Walsh, Patrick S; Allais, Florent; Zwier, Timothy S

    2014-10-22

    Ultraviolet spectroscopy of sinapoyl malate, an essential UV-B screening agent in plants, was carried out in the cold, isolated environment of a supersonic expansion to explore its intrinsic UV spectral properties in detail. Despite these conditions, sinapoyl malate displays anomalous spectral broadening extending well over 1000 cm(-1) in the UV-B region, presenting the tantalizing prospect that nature's selection of UV-B sunscreen is based in part on the inherent quantum mechanical features of its excited states. Jet-cooling provides an ideal setting in which to explore this topic, where complications from intermolecular interactions are eliminated. In order to better understand the structural causes of this behavior, the UV spectroscopy of a series of sinapate esters was undertaken and compared with ab initio calculations, starting with the simplest sinapate chromophore sinapic acid, and building up the ester side chain to sinapoyl malate. This "deconstruction" approach provided insight into the active mechanism intrinsic to sinapoyl malate, which is tentatively attributed to mixing of the bright V ((1)ππ*) state with an adiabatically lower (1)nπ* state which, according to calculations, shows unique charge-transfer characteristics brought on by the electron-rich malate side chain. All members of the series absorb strongly in the UV-B region, but significant differences emerge in the appearance of the spectrum among the series, with derivatives most closely associated with sinapoyl malate showing characteristic broadening even under jet-cooled conditions. The long vibronic progressions, conformational distribution, and large oscillator strength of the V (ππ*) transition in sinapates makes them ideal candidates for their role as UV-B screening agents in plants. PMID:25295994

  3. The lncRNA MALAT1, acting through HIF-1α stabilization, enhances arsenite-induced glycolysis in human hepatic L-02 cells.

    PubMed

    Luo, Fei; Liu, Xinlu; Ling, Min; Lu, Lu; Shi, Le; Lu, Xiaolin; Li, Jun; Zhang, Aihua; Liu, Qizhan

    2016-09-01

    Accelerated glycolysis, a common process in tumor cells called the Warburg effect, is associated with various biological phenomena. However, the role of glycolysis induced by arsenite, a well-established human carcinogen, is unknown. Long non-coding RNAs (lncRNAs) act as regulators in various cancers, but how lncRNAs regulate glucose metabolism remains largely unexplored. We have found that, in human hepatic epithelial (L-02) cells, arsenite increases lactate production; glucose consumption; and expression of glycolysis-related genes, including HK-2, Eno-1, and Glut-4. In L-02 cells exposed to arsenite, the lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and hypoxia inducible factors (HIFs)-α, the transcriptional regulators of cellular response to hypoxia, are over-expressed. In addition, HIF-1α, not HIF-2α, is involved in arsenite-induced glycolysis, and MALAT1 enhances arsenite-induced glycolysis. Although MALAT1 regulates HIF-α and promotes arsenite-induced glycolysis, MALAT1 promotes glycolysis through HIF-1α, not HIF-2α. Moreover, arsenite-increased MALAT1 enhances the disassociation of Von Hippel-Lindau (VHL) tumor suppressor from HIF-1α, alleviating VHL-mediated ubiquitination of HIF-1α, which causes accumulation of HIF-1α. In sum, these findings indicate that MALAT1, acting through HIF-1α stabilization, is a mediator that enhances glycolysis induced by arsenite. These results provide a link between the induction of lncRNAs and the glycolysis in cells exposed to arsenite, and thus establish a previously unknown mechanism for arsenite-induced hepatotoxicity. PMID:27287256

  4. Metabolic regulation of phytoplasma malic enzyme and phosphotransacetylase supports the use of malate as an energy source in these plant pathogens.

    PubMed

    Saigo, Mariana; Golic, Adrián; Alvarez, Clarisa E; Andreo, Carlos S; Hogenhout, Saskia A; Mussi, María A; Drincovich, María F

    2014-12-01

    Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power [NAD(P)H] generation from malate in phytoplasmas. PMID:25294105

  5. The enzymic degradation of alkyl-substituted gentisates, maleates and malates.

    PubMed

    Hopper, D J; Chapman, P J; Dagley, S

    1971-03-01

    1. Cell-free extracts, prepared from a non-fluorescent Pseudomonas grown on m-cresol, oxidized gentisate and certain alkyl-substituted gentisates with the consumption of 1 mol of oxygen and the formation of 1 mol of pyruvate from 1 mol of substrate. 2. In addition to pyruvate, malate was formed from gentisate; citramalate was formed from 3-methylgentisate and 4-methylgentisate; 2,3-dimethylmalate was formed from 3,4-dimethylgentisate. 3. One enantiomer, d-(-)-citramalate, was formed enzymically from 3-methylgentisate, 4-methylgentisate and citraconate. l-(+)-Citramalate was formed from mesaconate by the same extracts. When examined as its dimethyl ester by gas-liquid chromatography, enzymically formed 2,3-dimethylmalate showed the same behaviour as one of the two racemates prepared from the synthetic compound. 4. Maleate, citraconate and 2,3-dimethylmaleate were rapidly hydrated by cell extracts, but ethylfumarate and 2,3-dimethylfumarate were not attacked. 5. Cell extracts oxidized 1,4-dihydroxy-2-naphthoate to give pyruvate and phthalate. 6. Alkylgentisates were oxidized by a gentisate oxygenase (EC 1.13.1.4) present in Pseudomonas 2,5. The ring-fission products were attacked by maleylpyruvase, but not by fumarylpyruvase, and their u.v.-absorption spectra were those expected for alkyl-substituted maleylpyruvates. 7. When supplemented with ATP, CoA, succinate and Mg(2+) ions, an enzyme system from cells grown with 2,5-xylenol formed pyruvate from d- but not from l-citramalate. Extracts from cells grown with dl-citramalate or with itaconate attacked both d- and l-citramalate; other alkylmalates were cleaved in similar fashion to give pyruvate or 2-oxobutyrate. 8. These results accord with a general sequence of reactions in which the benzene nucleus of an alkylgentisate is cleaved to give an alkyl-substituted maleylpyruvate. The ring-fission products are hydrolysed to give pyruvate, plus alkylmalic acids which then undergo aldol fissions, probably as their Co

  6. The effect of the anti-angiogenic drug sunitinib malate on the vascular architecture of oral squamous cell carcinoma.

    PubMed

    Bampi, ViníCius Faccin; Gomes, Carolina Ferreira; De Oliveira, Laura Beatriz Oliveira; Da Silva, Jefferson Luis Braga

    2014-04-01

    The effects of anti-angiogenic therapies in guiding tumor angioarchitecture prompted us to examine the modifications in the vascular network of the oral squamous cell carcinoma (SCC) produced by the multitargeted tyrosine kinase inhibitor sunitinib malate. Twelve Syrian hamsters had their right buccal pouches submitted to tumor induction with dimethylbenzanthracene and carbamide peroxide for 55 days. The animals were then divided into two groups of six animals each; group I was treated with sunitinib malate and group II (control) was remained untreated. After 4 weeks, the hamsters had their vascular networks casted by Mercox® resin and analyzed by scanning electron microscopy. The qualitative study of the vascular network of the control tumor-bearing pouches showed images of intussusception and sprouting angiogenesis, flattened blood vessels, abrupt variations in their diameter, and a tortuous course. The samples treated with sunitinib exhibited a qualitative reduction of the signs of vascular proliferation. In addition, these casts presented an attenuation of the morphological features observed in the untreated tumor-bearing pouches. Quantitative analysis demonstrated that the pouches treated with sunitinib did not show a decrease (P > 0.05) in the vascular diameter and intervessel distances when compared with the control group. The results of the present study suggest that sunitinib may act on the vascular network of oral SCC, normalizing the blood vessels. However, further experiments should be performed in order to determine a judicious dose of this anti-angiogenic therapy. PMID:24458724

  7. Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants.

    PubMed

    Nunes-Nesi, Adriano; Carrari, Fernando; Lytovchenko, Anna; Smith, Anna M O; Loureiro, Marcelo Ehlers; Ratcliffe, R George; Sweetlove, Lee J; Fernie, Alisdair R

    2005-02-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the mitochondrial malate dehydrogenase gene in the antisense orientation and exhibiting reduced activity of this isoform of malate dehydrogenase show enhanced photosynthetic activity and aerial growth under atmospheric conditions (360 ppm CO2). In comparison to wild-type plants, carbon dioxide assimilation rates and total plant dry matter were up to 11% and 19% enhanced in the transgenics, when assessed on a whole-plant basis. Accumulation of carbohydrates and redox-related compounds such as ascorbate was also markedly elevated in the transgenics. Also increased in the transgenic plants was the capacity to use L-galactono-lactone, the terminal precursor of ascorbate biosynthesis, as a respiratory substrate. Experiments in which ascorbate was fed to isolated leaf discs also resulted in increased rates of photosynthesis providing strong indication for an ascorbate-mediated link between the energy-generating processes of respiration and photosynthesis. This report thus shows that the repression of this mitochondrially localized enzyme improves both carbon assimilation and aerial growth in a crop species. PMID:15665243

  8. Regulation of malate dehydrogenase (mdh) gene expression in Escherichia coli in response to oxygen, carbon, and heme availability.

    PubMed

    Park, S J; Cotter, P A; Gunsalus, R P

    1995-11-01

    Malate dehydrogenase catalyzes the interconversion of malate and oxaloacetate. It participates as a member of the tricarboxylic acid cycle and the branched noncyclic pathways under aerobic and anaerobic cell growth conditions, respectively. To investigate how the mdh gene is expressed under these different conditions, an mdh-lacZ operon fusion was constructed and analyzed in vivo. The mdh-lacZ fusion was expressed about twofold higher under aerobic conditions than under anaerobic cell growth conditions on most media tested. This anaerobic response is modulated by the ArcA protein, which functions as a repressor of mdh gene expression under both aerobic and anaerobic conditions. In contrast, mutations in the fnr gene did not affect mdh gene expression. Interestingly, cells grown anaerobically with glycerol and trimethylamine N-oxide or fumarate showed higher levels of mdh expression than did cells that were grown aerobically. Depending on the type of carbon compound used for cell growth, mdh expression varied by 11-fold and 5-fold under aerobic and anaerobic conditions, respectively. While mdh transcription was shown to be inversely proportional to the cell growth rate, cellular heme limitation stimulated a fivefold increase in mdh gene expression. The mdh gene appears to be highly regulated to adapt to changing conditions of aerobic and anaerobic cell growth with various types of carbon substrates. PMID:7592446

  9. Posttranscriptional silencing of the lncRNA MALAT1 by miR-217 inhibits the epithelial-mesenchymal transition via enhancer of zeste homolog 2 in the malignant transformation of HBE cells induced by cigarette smoke extract.

    PubMed

    Lu, Lu; Luo, Fei; Liu, Yi; Liu, Xinlu; Shi, Le; Lu, Xiaolin; Liu, Qizhan

    2015-12-01

    Lung cancer is regarded as the leading cause of cancer-related deaths, and cigarette smoking is one of the strongest risk factors for the development of lung cancer. However, the mechanisms for cigarette smoke-induced lung carcinogenesis remain unclear. The present study investigated the effects of an miRNA (miR-217) on levels of an lncRNA (MALAT1) and examined the role of these factors in the epithelial-mesenchymal transition (EMT) induced by cigarette smoke extract (CSE) in human bronchial epithelial (HBE) cells. In these cells, CSE caused decreases of miR-217 levels and increases in lncRNA MALAT1 levels. Over-expression of miR-217 with a mimic attenuated the CSE-induced increase of MALAT1 levels, and reduction of miR-217 levels by an inhibitor enhanced expression of MALAT1. Moreover, the CSE-induced increase of MALAT1 expression was blocked by an miR-217 mimic, indicating that miR-217 negatively regulates MALAT1 expression. Knockdown of MALAT1 reversed CSE-induced increases of EZH2 (enhancer of zeste homolog 2) and H3K27me3 levels. In addition to the alteration from epithelial to spindle-like mesenchymal morphology, chronic exposure of HBE cells to CSE increased the levels of EZH2, H3K27me3, vimentin, and N-cadherin and decreased E-cadherin levels, effects that were reversed by MALAT1 siRNA or EZH2 siRNA. The results indicate that miR-217 regulation of EZH2/H3K27me3 via MALAT1 is involved in CSE-induced EMT and malignant transformation of HBE cells. The posttranscriptional silencing of MALAT1 by miR-217 provides a link, through EZH2, between ncRNAs and the EMT and establishes a mechanism for CSE-induced lung carcinogenesis. PMID:26415832

  10. A NOVEL RED CLOVER HYDROXYCINNAMOYL TRANSFERASE HAS ENZYMATIC ACTIVITIES CONSISTENT WITH A ROLE IN PHASALIC ACID [2-O-(CAFFEOYL)-L-MALATE] BIOSYNTHESIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenylpropanoid o-diphenols accumulate in tissues of many plants functioning as defensive molecules and antioxidants. Red clover leaves accumulate high levels of two o-diphenols, phasalic acid [2-O-(caffeoyl)-L-malate] and clovamide [N-(caffeoyl)-L-DOPA]. In red clover, post-harvest oxidation of the...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Effects of the dicarboxylic acids malate and fumarate on E. coli 0157:H7 and Salmonella enterica Typhimurium populations in pure culture and in mixed ruminal microorganism fermentations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The dicarboxylic organic acids malate and fumarate increase ruminal pH, reduce methane production, increase propionate and total VFA production, and reduce lactic acid accumulation in a manner similar to ionophores. These acids stimulate the ruminal bacterium Selenomonas ruminantium to ferment lact...

  13. Cancer–Osteoblast Interaction Reduces Sost Expression in Osteoblasts and Up-Regulates lncRNA MALAT1 in Prostate Cancer

    PubMed Central

    Sebastian, Aimy; Hum, Nicholas R.; Hudson, Bryan D.; Loots, Gabriela G.

    2015-01-01

    Dynamic interaction between prostate cancer and the bone microenvironment is a major contributor to metastasis of prostate cancer to bone. In this study, we utilized an in vitro co-culture model of PC3 prostate cancer cells and osteoblasts followed by microarray based gene expression profiling to identify previously unrecognized prostate cancer–bone microenvironment interactions. Factors secreted by PC3 cells resulted in the up-regulation of many genes in osteoblasts associated with bone metabolism and cancer metastasis, including Mmp13, Il-6 and Tgfb2, and down-regulation of Wnt inhibitor Sost. To determine whether altered Sost expression in the bone microenvironment has an effect on prostate cancer metastasis, we co-cultured PC3 cells with Sost knockout (SostKO) osteoblasts and wildtype (WT) osteoblasts and identified several genes differentially regulated between PC3-SostKO osteoblast co-cultures and PC3-WT osteoblast co-cultures. Co-culturing PC3 cells with WT osteoblasts up-regulated cancer-associated long noncoding RNA (lncRNA) MALAT1 in PC3 cells. MALAT1 expression was further enhanced when PC3 cells were co-cultured with SostKO osteoblasts and treatment with recombinant Sost down-regulated MALAT1 expression in these cells. Our results suggest that reduced Sost expression in the tumor microenvironment may promote bone metastasis by up-regulating MALAT1 in prostate cancer.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 (...

  15. Design and construction of a non-natural malate to 1,2,4-butanetriol pathway creates possibility to produce 1,2,4-butanetriol from glucose.

    PubMed

    Li, Xinghua; Cai, Zhen; Li, Yin; Zhang, Yanping

    2014-01-01

    1,2,4-butanetriol (BT) is an important bulk chemical mainly used for producing the superior energetic plasticizer (1,2,4-butanetriol trinitrate) in propellant and explosive formulations. BT is commercially produced by chemical synthesis from petroleum-based feedstocks; until recently a costly biosynthetic route from xylose or arabinose was reported. Here we designed a novel biosynthetic pathway for BT from malate, for the purpose of using glucose as an alternative and cheaper substrate in future. This biosynthetic pathway was achieved through six sequential enzymatic reactions. Following tests of several combinations of enzymes for the pathway, five enzymes including malate thiokinase, succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate CoA-transferase and bifunctional aldehyde/alcohol dehydrogenase were finally chosen. All enzyme genes were expressed on two compatible plasmids in E. coli, and their functions verified separately. Following assembly of two functional modules, BT was detected in the fermentation broth upon addition of malate, proving BT can be biosynthesized from malate. Furthermore, BT was detected in the fermentation using glucose as the sole carbon source, suggesting that such novel BT biosynthetic pathway has created the possibility for the production of BT from the cheaper substrate glucose. PMID:25008973

  16. 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

  17. The Long Noncoding RNA MALAT-1 is A Novel Biomarker in Various Cancers: A Meta-analysis Based on the GEO Database and Literature

    PubMed Central

    Wang, Yumin; Xue, Dan; Li, Yuwei; Pan, Xuya; Zhang, Xueying; Kuang, Biao; Zhou, Ming; Li, Xiaoling; Xiong, Wei; Li, Guiyuan; Zeng, Zhaoyang; Yang, Tubao

    2016-01-01

    Background: MALAT-1 is significantly overexpressed in various cancers, suggesting that it might be a potential biomarker of cancer. Methods: A meta-analysis was performed using microarray data obtained via the Affymetrix Human Genome U133 Plus 2.0 platform found in the Gene Expression Omnibus (GEO) database and data obtained through a systematic search of PubMed and Web of Science. The pooled odds ratio (OR) and hazard ratio (HR) with 95% CI (Confidence interval) were used to judge the value of biomarkers. Results: A total of 28 studies were included in this meta-analysis, comprising a total of 3573 patients. MALAT-1 was significantly linked with over survival (OS) (HR=1.58, 95%CI: 1.12-2.23), recurrence-free survival (RFS) (HR=2.32, 95% CI: 1.68-3.19) and death-free survival (DFS) (HR=3.28, 95% CI: 1.52-7.09). We found that MALAT-1 was a risk factor in the prognoses of lung cancer (HR=1.54, 95%CI: 1.01-2.34), digestive system cancer (HR=2.16, 95% CI: 1.34-3.48) and ovarian cancer (HR=3.98, 95% CI: 1.54-10.25). In contrast, MALAT-1 was a safe factor in the prognosis of B cell lineage cancer (HR=0.45, 95% CI: 0.33-0.61). MALAT-1 was also a risk factor of RFS in breast cancer (HR=1.97, 95% CI: 1.25-3.09) and the TNM stage in pancreatic cancer (OR=3.65, 95% CI: 1.86-7.18) and glioma (OR=4.30, 95% CI: 1.90-9.73) and was a safe factor in colorectal cancer (OR=0.17, 95% CI: 0.08-0.35). MALAT-1 was significantly associated with lymph node metastasis in clear cell carcinoma (OR=5.04, 95% CI: 2.36-10.78) and distant metastasis in pancreatic cancer (OR=11.64, 95% CI: 2.13-63.78). Conclusions: MALAT-1 can serve as a molecular marker in different types of cancers. PMID:27313790

  18. Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)

    PubMed Central

    2005-01-01

    Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the Km values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate Km values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The Kact values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme. PMID:15989682

  19. Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

    PubMed

    Friedmann, Silke; Steindorf, Astrid; Alber, Birgit E; Fuchs, Georg

    2006-04-01

    The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle. PMID:16547052

  20. 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. PMID:26852108

  1. Unidirectional growth of potassium hydrogen malate single crystals and its characterizations on optical, mechanical, dielectric, laser damage threshold studies

    NASA Astrophysics Data System (ADS)

    Boopathi, K.; Rajesh, P.; Ramasamy, P.

    2013-02-01

    Single crystals of potassium hydrogen malate (PHM) were successfully grown by Sankaranarayanan-Ramasamy (SR) method and conventional slow evaporation solution technique which have the sizes of 35 mm in length, 20 mm in diameter and 15 × 10 × 3 mm3 respectively. The grown PHM crystals have been subjected to single crystal X-ray diffractometer, UV-Vis NIR studies, dielectric measurements, Vickers microhardness analysis and Laser damage threshold. The range and percentage of optical transmission is represented by recording UV-Vis-NIR analysis. The dielectric constant and loss measurement was made as function of temperature in the range of 40-150°C. Mechanical strength and laser stability of the SR method grown crystals was higher than the conventional method grown crystal.

  2. An investigation of the thermal stabilities of two malate dehydrogenases by comparison of their three-dimensional structures.

    PubMed

    Duffield, M L; Nicholls, D J; Atkinson, T; Scawen, M D

    1994-03-01

    The tertiary structure of Thermus aquaticus malate dehydrogenase (MDH) was predicted based on the known crystal structure of pig heart cytosolic MDH. Guanidinium chloride (GdmCl) unfolding experiments showed that there is only about a 4.2-kjoule/mol difference in delta G 0 between the pig and Thermus MDH. However, the two enzymes varied greatly in their [GdmCl]1/2, with Thermus MDH showing the expected increased stability (3.20 M against 0.58 M for pig MDH). The half-lives were determined for both Thermus MDH (34 min at 90 degrees C) and pig MDH (1.8 min at 60 degrees C). The Thermus MDH model was then examined to see what effect the substituted residues and changes may have on the enzyme, particularly in relation to its high thermal stability. PMID:8011596

  3. Structure-guided Discovery of Phenyl diketo-acids as Potent Inhibitors of M. tuberculosis Malate Synthase

    PubMed Central

    Krieger, Inna V.; Freundlich, Joel S.; Gawandi, Vijay B.; Roberts, Justin P.; Gawandi, Vidyadhar B.; Sun, Qingan; Owen, Joshua L.; Fraile, Maria T.; Huss, Sofia I.; Lavandera, Jose-Luis; Ioerger, Thomas R.; Sacchettini, James C.

    2012-01-01

    Summary The glyoxylate shunt plays an important role in fatty-acid metabolism, and has been shown to be critical to survival of several pathogens involved in chronic infections. For Mycobacterium tuberculosis (Mtb), a strain with a defective glyoxylate shunt was previously shown to be unable to establish infection in a mouse model. We report the development of novel phenyl-diketo acid (PDKA) inhibitors of malate synthase (GlcB), one of two glyoxylate shunt enzymes, using structure-based methods. PDKA inhibitors were active against Mtb grown on acetate, and over-expression of GlcB ameliorated this inhibition. Crystal structures of complexes of GlcB with PDKA inhibitors were used to guide optimization of potency. A selected PDKA compound demonstrated efficacy in a mouse model of tuberculosis. The discovery of these PDKA derivatives provides chemical validation of GlcB as an attractive target for tuberculosis therapeutics. PMID:23261599

  4. Features of structural organization and expression regulation of malate dehydrogenase isoforms from Rhodobacter sphaeroides strain 2R.

    PubMed

    Eprintsev, A T; Klimova, M A; Shikhalieva, K D; Fedorin, D N; Dzhaber, M T; Kompantseva, E I

    2009-07-01

    Two isoforms of malate dehydrogenase (MDH), dimeric and tetrameric, have been found in the purple non-sulfur bacterium Rhodobacter sphaeroides strain 2R, devoid of the glyoxylate shunt, which assimilate acetate via the citramalate cycle. Inhibitory analysis showed that the 74-kDa protein is involved in tricarboxylic acid cycle, while the 148-kDa MDH takes part in the citramalate pathway. A single gene encoding synthesis of the isologous subunits of the MDH isoforms was found during molecular-biological investigations. The appearance in the studied bacterium of the tetrameric MDH isoform during growth in the presence of acetate is probably due to the increased level of mdh gene expression, revealed by the real-time PCR, the product of which in cooperation with the citramalate cycle enzymes plays an important role in acetate assimilation. PMID:19747101

  5. Loss of Mitochondrial Malate Dehydrogenase Activity Alters Seed Metabolism Impairing Seed Maturation and Post-Germination Growth in Arabidopsis.

    PubMed

    Sew, Yun Shin; Ströher, Elke; Fenske, Ricarda; Millar, A Harvey

    2016-06-01

    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

  6. Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling.

    PubMed

    Lindén, Pernilla; Keech, Olivier; Stenlund, Hans; Gardeström, Per; Moritz, Thomas

    2016-05-01

    Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a (13)CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1 The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and (13)C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions. PMID:26889011

  7. Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling

    PubMed Central

    Lindén, Pernilla; Keech, Olivier; Stenlund, Hans; Gardeström, Per; Moritz, Thomas

    2016-01-01

    Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a 13CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1. The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and 13C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions. PMID:26889011

  8. Crystal Structures and Molecular Dynamics Simulations of Thermophilic Malate Dehydrogenase Reveal Critical Loop Motion for Co-Substrate Binding

    PubMed Central

    Luo, Huei-Ru; Wu, Szu-Pei; Hsu, Chun-Hua

    2013-01-01

    Malate dehydrogenase (MDH) catalyzes the conversion of oxaloacetate and malate by using the NAD/NADH coenzyme system. The system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. We elucidated the biochemical and structural features of MDH from Thermus thermophilus (TtMDH) for use in various biotechnological applications. The biochemical characterization of recombinant TtMDH revealed greatly increased activity above 60°C and specific activity of about 2,600 U/mg with optimal temperature of 90°C. Analysis of crystal structures of apo and NAD-bound forms of TtMDH revealed a slight movement of the binding loop and few structural elements around the co-substrate binding packet in the presence of NAD. The overall structures did not change much and retained all related positions, which agrees with the CD analyses. Further molecular dynamics (MD) simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. Interestingly, at the simulated structure of 353 K, a large change occurred around the active site such that with increasing temperature, a mobile loop was closed to co-substrate binding region. From biochemical characterization, structural comparison and MD simulations, the thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzyme's activity. PMID:24386145

  9. Substrate-dependent utilization of the glycerol 3-phosphate or malate/aspartate redox shuttles by Ehrlich ascites cells.

    PubMed Central

    Grivell, A R; Korpelainen, E I; Williams, C J; Berry, M N

    1995-01-01

    The rate of transfer of reducing equivalents from cytoplasm to mitochondria has been examined in Ehrlich ascites tumour cells incubated in the presence of lactate. The flux of reducing equivalents was determined from the rate of metabolism of reduced intermediates that are oxidized within the cytosol. The magnitude of the flux of reducing equivalents was dependent on both the concentration of added lactate and the presence of carbohydrate. The rate of flux was twice as great in the presence of glucose and four times as high when glucose and lactate were added together as when lactate was the only added substrate. Fructose was less effective than glucose in stimulating reducing equivalent flux. In the presence of glucose or fructose, there was a substantial accumulation of hexose phosphates, dihydroxyacetone phosphate and glycerol 3-phosphate. Rotenone, an inhibitor of NADH dehydrogenase, and amino-oxyacetate, which inhibits the malate/aspartate shuttle, were powerful suppressors of reducing equivalent flux from lactate as sole substrate, but were much less potent in the presence of carbohydrate. Antimycin substantially inhibited reducing equivalent flux from all combinations of added substrates, consistent with its ability to block oxidation of reducing equivalents transferred by both the malate/aspartate and glycerol 3-phosphate shuttles. The glycerol 3-phosphate shuttle represents around 80% of the maximum total observed activity but is active only while glycolytic intermediates are present to provide the necessary substrates of the shuttle. This Ehrlich ascites cell line has an essentially similar total reducing equivalent shuttle capacity to that of isolated hepatocytes. PMID:7654209

  10. CHARACTERIZATION OF THE N-ACETYL-α-D-GLUCOSAMINYL L-MALATE SYNTHASE (BshA) AND DEACETYLASE (BshB) FUNCTIONS FOR BACILLITHIOL BIOSYNTHESIS IN Bacillus anthracis†‡

    PubMed Central

    Parsonage, Derek; Newton, Gerald L.; Holder, Robert C.; Wallace, Bret D.; Paige, Carleitta; Hamilton, Chris J.; Dos Santos, Patricia C.; Redinbo, Matthew R.; Reid, Sean D.; Claiborne, Al

    2010-01-01

    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 α-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 (→GlcNAc-malate) and the BaBshB deacetylase (→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 ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of L-malate, but not other α-hydroxyacids, as 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, suggesting that the biosynthesis of BSH could represent a target for 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. PMID:20799687

  11. Effects of essential oils, yeast culture and malate on rumen fermentation, blood metabolites, growth performance and nutrient digestibility of Baluchi lambs fed high-concentrate diets.

    PubMed

    Malekkhahi, M; Tahmasbi, A M; Naserian, A A; Danesh Mesgaran, M; Kleen, J L; Parand, A A

    2015-04-01

    The experiment was conducted to evaluate the effects of dietary supplementation with a mixture of essential oils (MEO), yeast culture (YC) and malate on performance, nutrient digestion, rumen fermentation and blood metabolites of lambs fed high-concentrate growing diets. For this purpose, twenty Baluchi lambs (17.3 ± 0.5 kg body weight and 3 months old) were randomly assigned to four dietary treatments in a completely randomized design with five lambs per treatment. The treatment groups were as follows: (i) control: basal diet without any additive, (ii) basal diet plus 400 mg/day MEO (thymol, carvacrol, eugenol, limonene and cinnamaldehyde), (iii) basal diet with 4 g/day YC and (iv) basal diet plus 4 g/day malate. No differences between the dietary treatments were observed in dry matter intake, average daily gain or feed conversion ratio (p > 0.05). Compared with control and malate treatment, lambs fed MEO and YC had an improved crude protein digestibility (p < 0.05). Yeast culture significantly increased (p > 0.05) cell wall digestibility compared to the other treatments. No differences were observed between treatments with respect to nitrogen balance or ruminal pH and ammonia concentrations (p > 0.05). No differences were observed between treatments with respect to ruminal total volatile fatty acid concentration and molar proportions of acetate, butyrate and valerate. Molar proportion of propionate was higher (p < 0.05) for YC and malate compared to control and MEO. Plasma glucose concentration was higher (p < 0.05) in lambs fed YC and malate than in lambs fed the control or the MEO diet. Blood concentration of triglycerides significantly decreased when feeding the MEO and YC diets (p < 0.05). It was concluded that YC may be more useful as a feed additive for manipulation of rumen fermentation in lambs fed with high-concentrate diets than MEO and malate, because YC enhanced crude protein and cell wall digestibility, ruminal molar proportion

  12. Effect of Shoot Removal and Malate on the Activity of Nitrate Reductase Assayed in Vivo in Barley Roots (Hordeum vulgare cv. Midas)

    PubMed Central

    Deane-Drummond, Celia E.; Clarkson, David T.; Johnson, Christopher B.

    1979-01-01

    There is a diurnal variation of nitrate reductase activity (NRA) measured in vivo in barley roots (Hordeum vulgare cv. Midas). In intact plants receiving a 16-hour photoperiod, NRA increases when the light is switched on, reaches a maximum value after 7 to 8 hours, and thereafter declines. Shoot removal (detopping) at the start of the photoperiod prevents the rise in NRA; detopping after 5 hours light leads to a rapid fall in NRA. The inclusion of 10 millimolar malate in the external medium causes a rise in NRA in plants detopped at the beginning of the photoperiod and thus seems to substitute partially for the illuminated shoot. Oxalate, fumarate, and tartrate did not have this effect. Preincubation of the roots of intact plants with 10 millimolar malate for 3 hours, prior to detopping, causes an increase in the flux of amino acids into the xylem sap of detopped roots. PMID:16661028

  13. Crystallization and preliminary X-ray diffraction studies of tetrameric malate dehydrogenase from the novel Antarctic psychrophile Flavobacterium frigidimaris KUC-1

    SciTech Connect

    Fujii, Tomomi; Oikawa, Tadao; Muraoka, Ikuo; Soda, Kenji; Hata, Yasuo

    2007-11-01

    A psychrophilic malate dehydrogenase from the novel Antarctic bacterium F. frigidimaris KUC-1 was crystallized using the hanging-drop vapour-diffusion method. The crystals contained one tetrameric molecule per asymmetric unit. The best crystal diffracted to 1.8 Å resolution. Flavobacterium frigidimaris KUC-1 is a novel psychrotolerant bacterium isolated from Antarctic seawater. Malate dehydrogenase (MDH) is an essential metabolic enzyme in the citric acid cycle and has been cloned, overexpressed and purified from F. frigidimaris KUC-1. In contrast to the already known dimeric form of MDH from the psychrophile Aquaspirillium arcticum, F. frigidimaris MDH exists as a tetramer. It was crystallized at 288 K by the hanging-drop vapour-diffusion method using ammonium sulfate as the precipitating agent. The crystal diffracted to a maximum resolution of 1.80 Å. It contains one tetrameric molecule in the asymmetric unit.

  14. Structure and function of Plasmodium falciparum malate dehydrogenase: role of critical amino acids in co-substrate binding pocket.

    PubMed

    Pradhan, Anupam; Tripathi, Abhai K; Desai, Prashant V; Mukherjee, Prasenjit K; Avery, Mitchell A; Walker, Larry A; Tekwani, Babu L

    2009-01-01

    The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory 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 glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co

  15. Isolation of the facA (acetyl-coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans.

    PubMed

    Sandeman, R A; Hynes, M J

    1989-07-01

    Acetate inducible genes of Aspergillus nidulans were cloned via differential hybridization to cDNA probes. Using transformation of mutant strains the genes were identified as facA (acetyl-Coenzyme A synthetase) and acuE (malate synthase). The levels of RNA encoded by these genes were shown to be acetate inducible and subject to carbon catabolite repression. Induction is abolished in a facB mutant and carbon catabolite repression is relieved in a creA mutant. PMID:2571070

  16. The mitochondrial malate dehydrogenase 1 gene GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton.

    PubMed

    Wang, Zhi-An; Li, Qing; Ge, Xiao-Yang; Yang, Chun-Lin; Luo, Xiao-Li; Zhang, An-Hong; Xiao, Juan-Li; Tian, Ying-Chuan; Xia, Gui-Xian; Chen, Xiao-Ying; Li, Fu-Guang; Wu, Jia-He

    2015-01-01

    Cotton, an important commercial crop, is cultivated for its natural fibers, and requires an adequate supply of soil nutrients, including phosphorus, for its growth. Soil phosporus exists primarily in insoluble forms. We isolated a mitochondrial malate dehydrogenase (MDH) gene, designated as GhmMDH1, from Gossypium hirsutum L. to assess its effect in enhancing P availability and absorption. An enzyme kinetic assay showed that the recombinant GhmMDH1 possesses the capacity to catalyze the interconversion of oxaloacetate and malate. The malate contents in the roots, leaves and root exudates was significantly higher in GhmMDH1-overexpressing plants and lower in knockdown plants compared with the wild-type control. Knockdown of GhmMDH1 gene resulted in increased respiration rate and reduced biomass whilst overexpression of GhmMDH1 gave rise to decreased respiration rate and higher biomass in the transgenic plants. When cultured in medium containing only insoluble phosphorus, Al-phosphorus, Fe-phosphorus, or Ca-phosphorus, GhmMDH1-overexpressing plants produced significantly longer roots and had a higher biomass and P content than WT plants, however, knockdown plants showed the opposite results for these traits. Collectively, our results show that GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton, owing to its functions in leaf respiration and P acquisition. PMID:26179843

  17. The mitochondrial malate dehydrogenase 1 gene GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton

    PubMed Central

    Wang, Zhi-An; Li, Qing; Ge, Xiao-Yang; Yang, Chun-Lin; Luo, Xiao-Li; Zhang, An-Hong; Xiao, Juan-Li; Tian, Ying-Chuan; Xia, Gui-Xian; Chen, Xiao-Ying; Li, Fu-Guang; Wu, Jia-He

    2015-01-01

    Cotton, an important commercial crop, is cultivated for its natural fibers, and requires an adequate supply of soil nutrients, including phosphorus, for its growth. Soil phosporus exists primarily in insoluble forms. We isolated a mitochondrial malate dehydrogenase (MDH) gene, designated as GhmMDH1, from Gossypium hirsutum L. to assess its effect in enhancing P availability and absorption. An enzyme kinetic assay showed that the recombinant GhmMDH1 possesses the capacity to catalyze the interconversion of oxaloacetate and malate. The malate contents in the roots, leaves and root exudates was significantly higher in GhmMDH1-overexpressing plants and lower in knockdown plants compared with the wild-type control. Knockdown of GhmMDH1 gene resulted in increased respiration rate and reduced biomass whilst overexpression of GhmMDH1 gave rise to decreased respiration rate and higher biomass in the transgenic plants. When cultured in medium containing only insoluble phosphorus, Al-phosphorus, Fe-phosphorus, or Ca-phosphorus, GhmMDH1-overexpressing plants produced significantly longer roots and had a higher biomass and P content than WT plants, however, knockdown plants showed the opposite results for these traits. Collectively, our results show that GhmMDH1 is involved in plant and root growth under phosphorus deficiency conditions in cotton, owing to its functions in leaf respiration and P acquisition. PMID:26179843

  18. Purification and characterization of recombinant malate synthase enzymes from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585.

    PubMed

    Loke, P; Goh, L L; Seng Soh, B; Yeow, P; Sim, T S

    2002-04-01

    Malate synthases (MS) from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585 were cloned by polymerase chain reaction into a glutathione S-transferase (GST) fusion expression vector and heterologously expressed in Escherichia coli. The fusion GST-MS construct improved the soluble expression of MS by approximately 10-fold compared to the soluble expression of nonfusion MS. With the significant improvement in levels of soluble MS, purification and subsequent cleavage of recombinant MS from GST were facilitated in this study. Using purified enzymes, optimized parameters, which achieved maximal specific activity, were established in the enzymatic assay for streptomycete MS. The average purified specific activities of S. coelicolor and S. clavuligerus MS were 26199 and 11821 nmol/mg min, respectively. Furthermore, enzymatic analysis revealed that the two streptomycete MS displayed a similar Km value for acetyl-CoA, but S. coelicolor MS had a Km value for glyoxylate that is approximately sixfold higher than S. clavuligerus MS. PMID:11986926

  19. Glyoxysomal malate dehydrogenase in pumpkin: cloning of a cDNA and functional analysis of its presequence.

    PubMed

    Kato, A; Takeda-Yoshikawa, Y; Hayashi, M; Kondo, M; Hara-Nishimura, I; Nishimura, M

    1998-02-01

    Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of the glyoxylate cycle that participates in degradation of storage oil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledons that encodes a polypeptide consisting of 356 amino acid residues. The nucleotide and N-terminal amino acid sequences revealed that gMDH is synthesized as a precursor with an N-terminal extrapeptide. The N-terminal presequence of 36 amino acid residues contains two regions homologous to those of other microbody proteins, which are also synthesized as large precursors. To investigate the functions of the N-terminal presequence of gMDH, we generated transgenic Arabidopsis that expressed a chimeric protein consisting of beta-glucuronidase and the N-terminal region of gMDH. Immunological and immunocytochemical studies revealed that the chimeric protein was imported into microbodies such as glyoxysomes and leaf peroxisomes and was then subsequently processed. Site-directed mutagenesis studies showed that the conserved amino acids in the N-terminal presequence, Arg-10 and His-17, function as recognition sites for the targeting to plant microbodies, and Cys-36 in the presequence is responsible for its processing. These results correspond to those from the analyses of glyoxysomal citrate synthase (gCS), which was also synthesized as a large precursor, suggesting that common mechanisms that can recognize the targeting or the processing of gMDH and gCS function in higher plant cells. PMID:9559562

  20. A validated reversed phase HPLC method for the determination of process-related impurities in almotriptan malate API.

    PubMed

    Kumar, A Phani; Ganesh, V R L; Rao, D V Subba; Anil, C; Rao, B Venugopala; Hariharakrishnan, V S; Suneetha, A; Sundar, B Syama

    2008-03-13

    An isocratic reversed phase liquid chromatographic (RP-LC) method has been developed and subsequently validated for the determination of almotriptan malate and its process-related impurities. Separation was achieved with a Phenomenex, Gemini, C-18 column and sodium phosphate buffer (pH adjusted to 7.6): acetonitrile (80:20, v/v) as eluent, at a flow rate of 1.5 mL/min. UV detection was performed at 227 nm. The method is simple, rapid, selective, accurate and stability indicating. The described method is linear over a range of LOQ, 1.5 ug/mL (150% of the specification limit) for all the process-related impurities. The method precision for the determination of related compounds was below 1.0% R.S.D. The accuracy of the method demonstrated at 4 levels in the range of 25-150% of the specification limit and the recovery of impurities were found to be in the range of 96-102%. The method is useful in the quality control of bulk manufacturing. PMID:18191357

  1. Up-regulation of the mitochondrial malate dehydrogenase by oxidative stress is mediated by miR-743a

    PubMed Central

    Shi, Qingli; Gibson, Gary E.

    2011-01-01

    These experiments reveal for the first time that microRNAs 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 (AD). Oxidative stress, an early and pervasive event in AD, 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 microRNAs regulate gene expression post-transcriptionally, the expression of miR-743a, a microRNA 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 microRNA in oxidative stress and neurodegeneration. PMID:21623795

  2. Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle: Implications for Neuronal Energy Metabolism.

    PubMed

    Satrústegui, Jorgina; Bak, Lasse K

    2015-12-01

    The malate-aspartate NADH shuttle (MAS) operates in neurons and other cells to translocate reducing equivalents from the cytosol to the mitochondrial matrix, thus allowing a continued flux through the glycolytic pathway and metabolism of extracellular lactate. Recent discoveries have taught us that MAS is regulated by fluctuations in cytosolic Ca(2+) levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca(2+) fluctuations below the threshold of the mitochondrial calcium uniporter, there is a positive correlation between Ca(2+) and MAS activity; however, if cytosolic Ca(2+) increases above the threshold, MAS activity is thought to be reduced by an intricate mechanism. The latter forces the neurons to partly rely on anaerobic glycolysis producing lactate that may be metabolized subsequently, by neurons or other cells. In this review, we will discuss the evidence for Ca(2+)-mediated regulation of MAS that have been uncovered over the last decade or so, together with the need for further verification, and examine the metabolic ramifications for neurons. PMID:26138554

  3. Sunitinib malate (SU-11248) reduces tumour burden and lung metastasis in an intratibial human xenograft osteosarcoma mouse model

    PubMed Central

    Kumar, Ram Mohan Ram; Arlt, Matthias JE; Kuzmanov, Aleksandar; Born, Walter; Fuchs, Bruno

    2015-01-01

    Osteosarcoma is a rare type of cancer that commonly occurs as a primary bone tumour in children and adolescents and is associated with a poor clinical outcome. Despite complex treatment protocols, including chemotherapy combined with surgical resection, the prognosis for patients with osteosarcoma and metastases remains poor and more effective therapies are required. In this study, we evaluated the therapeutic efficacy of sunitinib malate, a wide-spectrum tyrosine kinase inhibitor, in a preclinical mouse model of osteosarcoma. Sunitinib significantly inhibited proliferation, provoked apoptosis and induced G2/M cell cycle arrest in the human osteosarcoma cell lines SaOS-2 and 143B in vitro. Importantly, sunitinib treatment significantly reduced tumour burden, microvessel density and suppressed pulmonary metastasis in a 143B cell-derived intratibial osteosarcoma model in SCID mice. Sunitinib significantly decreased primary tumor tissue proliferation and reduced tumor vasculature. Our study indicates that sunitinib has potential for effective treatment of metastasizing osteosarcoma and provides the framework for future clinical trials with sunitinib alone or in combination with conventional and other novel therapeutics aiming at increased treatment efficacy and improved patient outcome. PMID:26328246

  4. Implication of citrate, malate and histidine in the accumulation and transport of nickel in Mesembryanthemum crystallinum and Brassica juncea.

    PubMed

    Amari, Taoufik; Lutts, Stanley; Taamali, Manel; Lucchini, Giorgio; Sacchi, Gian Attilio; Abdelly, Chedly; Ghnaya, Tahar

    2016-04-01

    Citrate, malate and histidine have been involved in many processes including metal tolerance and accumulation in plants. These molecules have been frequently reported to be the potential nickel chelators, which most likely facilitate metal transport through xylem. In this context, we assess here, the relationship between organics acids and histidine content and nickel accumulation in Mesembryanthemum crystallinum and Brassica juncea grown in hydroponic media added with 25, 50 and 100 µM NiCl2. Results showed that M. crystallinum is relatively more tolerant to Ni toxicity than B. juncea. For both species, xylem transport rate of Ni increased with increasing Ni supply. A positive correlation was established between nickel and citrate concentrations in the xylem sap. In the shoot of B. juncea, citric and malic acids concentrations were significantly higher than in the shoot of M. crystallinum. Also, the shoots and roots of B. juncea accumulated much more histidine. In contrast, a higher root citrate concentration was observed in M. crystallinum. These findings suggest a specific involvement of malic and citric acid in Ni translocation and accumulation in M. crystallinum and B. juncea. The high citrate and histidine accumulation especially at 100µM NiCl2, in the roots of M. crystallinum might be among the important factors associated with the tolerance of this halophyte to toxic Ni levels. PMID:26745003

  5. 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. PMID:19129175

  6. 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. PMID:26946085

  7. Improved Production of Propionic Acid in Propionibacterium jensenii via Combinational Overexpression of Glycerol Dehydrogenase and Malate Dehydrogenase from Klebsiella pneumoniae

    PubMed Central

    Liu, Long; Zhuge, Xin; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua

    2015-01-01

    Microbial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineering Propionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis in P. jensenii by examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-type P. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-type P. jensenii to 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction in P. jensenii and further improved PA titers via the coexpression of GDH and MDH, making the engineered P. jensenii strain a potential industrial producer of PA. PMID:25595755

  8. NAD-dependent malate dehydrogenase protects against oxidative damage in Escherichia coli K-12 through the action of oxaloacetate.

    PubMed

    Oh, Tae Jeong; Kim, In Gyu; Park, Seon Young; Kim, Kug Chan; Shim, Hye Won

    2002-01-01

    Reactive oxygen species including hydrogen peroxide (H(2)O(2)) and hydroxyl radical (OH) can be generated by ionizing radiation and has the potential to induce diseases. We provide the evidence that NAD-dependent malate dehydrogenase (MDH) is involved in the antioxidant role in preventing H(2)O(2) or γ-radiation-induced damage in Escherichia coli through the action of oxaloacetate. The E. colimdh mutant strain defective in MDH activity was more sensitive to H(2)O(2) or γ-radiation than was the wild type strain, when challenged in the exponential growth phase. The mdh mutant cells pretreated with oxaloacetate (2.5 mM), a product of NAD-dependent MDH activity, prior to H(2)O(2) treatment or γ-irradiation are resistant to H(2)O(2) or γ-radiation-induced damage, so cell survivability is restored to similar levels with the wild type. The SOS induction of umu'-'lacZ fusion gene by H(2)O(2) is significantly repressed by pretreatment of oxaloacetate in a dose-dependent way. These results indicate that oxaloacetate effectively protects E. coli cells against damage caused by oxidative stress. Oxaloacetate strongly prevented the DNA strand breaks by OH in a metal-catalyzed oxidation (MCO) system that generated H(2)O(2) as a mediator. By contrast, the prevention of DNA damage by oxaloacetate in an γ-irradiation system that directly generates OH from H(2)O in vitro was far less than that in an MCO system. Our results demonstrated that oxaloacetate, metabolite of NAD-dependent MDH action, plays a role as an antioxidant, possibly by scavenging H(2)O(2). PMID:21782581

  9. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    SciTech Connect

    Li, Yongchao; Tschaplinski, Timothy J; Engle, Nancy L; Hamilton, Choo Yieng; Rodriguez, Jr., Miguel; Liao, James C; Schadt, Christopher Warren; Guss, Adam M; Yang, Yunfeng; Graham, David E

    2012-01-01

    Background: The model bacterium Clostridium cellulolyticum efficiently hydrolyzes crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels. Therefore genetic engineering will likely be required to improve the ethanol yield. Random mutagenesis, plasmid transformation, and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism. Results: The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products (by molarity), corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four-times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant s TCA pathway. Conclusions: The efficient intron-based gene inactivation system produced the first gene-targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to

  10. Toggle release

    NASA Technical Reports Server (NTRS)

    Graves, Thomas Joseph (Inventor); Yang, Robert Alexander (Inventor); Brown, Christopher William (Inventor)

    1988-01-01

    The invention relates to a pyrotechnic actuated release mechanism which is mechanically two fault tolerant for effecting release. It is particularly well suited for releasably connecting structures to be used in the space environment or in other aerospace applications. The device comprises a fastener plate and fastener body, each attachable to either one of a pair of structures to be joined. The fastener plate and the body are fastenable by a toggle supported at one end on the fastener plate and mounted for universal pivotal movement thereon. At its other end, which is received in a central opening in the fastener body and adapted for limited pivotal movement therein, the toggle is restrained by three retractable latching pins. Each pin is individually retractable by combustion of a pyrotechnic charge. While retraction of all three pins releases the toggle, the fastener is mechanically two fault tolerant since the failure of any single or pair of the latch pins to retract results in an asymmetrical loading on the toggle and its pivotal movement to effect a release. An annular bolt is mounted on the fastener plate as a support for the socket mounting of the toggle whereby its selective axial movement provides a means for pre-loading the toggle.