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Sample records for cytosolic sulfotransferases sult1

  1. Sulfation of ritodrine by the human cytosolic sulfotransferases (SULTs): Effects of SULT1A3 genetic polymorphism.

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    Hui, Ying; Liu, Ming-Cheh

    2015-08-15

    Previous studies have demonstrated the metabolism of ritodrine through sulfation. The current study was designed to identify the human SULTs that are capable of sulfating ritodrine and to investigate how genetic polymorphism of the major ritodrine-sulfating SULT, SULT1A3, may affect its sulfating activity. A systematic analysis revealed that of the 13 known human SULTs, SULT1A1, SULT1A3, and SULT1C4, were capable of mediating the sulfation of ritodrine, with SULT1A3 displaying the strongest sulfating activity. Effects of genetic polymorphism on the sulfating activity of SULT1A3 were examined. By employing site-directed mutagenesis, 4 SULT1A3 allozymes were generated, expressed, and purified. Purified SULT1A3 allozymes were shown to exhibit differential sulfating activity toward ritodrine. Kinetic studies further demonstrated differential substrate affinity and catalytic efficiency among the SULT1A3 allozymes. Collectively, these results provided useful information concerning the differential metabolism of ritodrine through sulfation in different individuals. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Sulfation of fulvestrant by human liver cytosols and recombinant SULT1A1 and SULT1E1

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    Edavana VK

    2011-11-01

    Full Text Available Vineetha Koroth Edavana1, Xinfeng Yu1, Ishwori B Dhakal1, Suzanne Williams1, Baitang Ning2, Ian T Cook3, David Caldwell1, Charles N Falany3, Susan Kadlubar11Division of Medical Genetics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA; 2Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, USA; 3Department of Pharmacology, University of Alabama, Birmingham, AL, USAAbstract: Fulvestrant (Faslodex™ is a pure antiestrogen that is approved to treat hormone receptor-positive metastatic breast cancer in postmenopausal women. Previous studies have demonstrated that fulvestrant metabolism in humans involves cytochromes P450 and UDP-glucuronosyltransferases (UGTs. To date, fulvestrant sulfation has not been characterized. This study examined fulvestrant sulfation with nine recombinant sulfotransferases and found that only SULT1A1 and SULT1E1 displayed catalytic activity toward this substrate, with Km of 4.2 ± 0.99 and 0.2 ± 0.16 µM, respectively. In vitro assays of 104 human liver cytosols revealed marked individual variability that was highly correlated with β-naphthol sulfation (SULT1A1 diagnostic substrate; r = 0.98, P < 0.0001, but not with 17ß-estradiol sulfation (SULT1E1 diagnostic substrate; r = 0.16, P = 0.10. Fulvestrant sulfation was correlated with both SULT1A1*1/2 genotype (P value = 0.023 and copy number (P < 0.0001. These studies suggest that factors influencing SULT1A1/1E1 tissue expression and/or enzymatic activity could influence the efficacy of fulvestrant therapy.Keywords: fulvestrant, sulfotransferase, genotype, copy number

  3. Crystal structures of human sulfotransferases SULT1B1 and SULT1C1 complexed with the cofactor product adenosine-3'- 5'-diphosphate (PAP)

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    Dombrovski, Luidmila; Dong, Aiping; Bochkarev, Alexey; Plotnikov, Alexander N. (Toronto)

    2008-09-17

    Cytosolic sulfotransferases (SULTs), often referred as Phase II enzymes of chemical defense, are a superfamily of enzymes that catalyze the transfer of a sulfonate group from 3{prime}-phosphoadenosine 5{prime}-phosphosulfate (PAPS) to an acceptor group of substrates. This reaction modulates the activities of a large array of small endogenous and foreign chemicals including drugs, toxic compounds, steroid hormones, and neurotransmitters. In some cases, however, SULTs activate certain food and environmental compounds to mutagenenic and carcinogenic metabolites. Twelve human SULTs have been identified, which are partitioned into three families: SULT1, SULT2 and SULT4. The SULT1 family is further divided in four subfamilies, A, B, C, and E, and comprises eight members (1A1, 1A2, 1A3, 1B1, 1C1, 1C2, 1C3, and 1E1). Despite sequence and structural similarity among the SULTs, the family and subfamily members appear to have different biological function. SULT1 family shows substrate-binding specificity for simple phenols, estradiol, and thyroid hormones, as well as environmental xenobiotics and drugs. Human SULT1B1 is expressed in liver, colon, small intestine, and blood leukocytes, and shows substrate-binding specificity to thyroid hormones and benzylic alcohols. Human SULT1C1 is expressed in the adult stomach, kidney, and thyroid, as well as in fetal kidney and liver. SULT1C1 catalyzes the sulfonation of p-nitrophenol and N-hydroxy-2-acetylaminofluorene in vitro. However, the in vivo function of the enzyme remains unknown. We intend to solve the structures for all of the SULTs for which structural information is not yet available, and compare the structural and functional features of the entire SULT superfamily. Here we report the structures of two members of SULT1 family, SULT1B1 and SULT1C1, both in complex with the product of the PAPS cofactor, adenosine-3{prime}-5{prime}-diphosphate (PAP).

  4. Sulfation of 6-Gingerol by the Human Cytosolic Sulfotransferases: A Systematic Analysis.

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    Luo, Lijun; Mei, Xue; Xi, Yuecheng; Zhou, Chunyang; Hui, Ying; Kurogi, Katsuhisa; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh

    2016-02-01

    Previous studies have demonstrated the presence of the sulfated form of 6-gingerol, a major pharmacologically active component of ginger, in plasma samples of normal human subjects who were administered 6-gingerol. The current study was designed to systematically identify the major human cytosolic sulfotransferase enzyme(s) capable of mediating the sulfation of 6-gingerol. Of the 13 known human cytosolic sulfotransferases examined, six (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C4, SULT1E1) displayed significant sulfating activity toward 6-gingerol. Kinetic parameters of SULT1A1, SULT1A3, SULT1C4, and SULT1E1 that showed stronger 6-gingerol-sulfating activity were determined. Of the four human organ samples tested, small intestine and liver cytosols displayed considerably higher 6-gingerol-sulfating activity than those of the lung and kidney. Moreover, sulfation of 6-gingerol was shown to occur in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under the metabolic setting. Collectively, these results provided useful information relevant to the metabolism of 6-gingerol through sulfation both in vitro and in vivo. Georg Thieme Verlag KG Stuttgart · New York.

  5. Sulfation of afimoxifene, endoxifen, raloxifene, and fulvestrant by the human cytosolic sulfotransferases (SULTs: A systematic analysis

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    Ying Hui

    2015-07-01

    Full Text Available Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity.

  6. Sulphation of acetaminophen by the human cytosolic sulfotransferases: a systematic analysis.

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    Yamamoto, Akihiro; Liu, Ming-Yih; Kurogi, Katsuhisa; Sakakibara, Yoichi; Saeki, Yuichi; Suiko, Masahito; Liu, Ming-Cheh

    2015-12-01

    Sulphation is known to be critically involved in the metabolism of acetaminophen in vivo. This study aimed to systematically identify the major human cytosolic sulfotransferase (SULT) enzyme(s) responsible for the sulphation of acetaminophen. A systematic analysis showed that three of the twelve human SULTs, SULT1A1, SULT1A3 and SULT1C4, displayed the strongest sulphating activity towards acetaminophen. The pH dependence of the sulphation of acetaminophen by each of these three SULTs was examined. Kinetic parameters of these three SULTs in catalysing acetaminophen sulphation were determined. Moreover, sulphation of acetaminophen was shown to occur in HepG2 human hepatoma cells and Caco-2 human intestinal epithelial cells under the metabolic setting. Of the four human organ samples tested, liver and intestine cytosols displayed considerably higher acetaminophen-sulphating activity than those of lung and kidney. Collectively, these results provided useful information concerning the biochemical basis underlying the metabolism of acetaminophen in vivo previously reported. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  7. Sulfation of afimoxifene, endoxifen, raloxifene, and fulvestrant by the human cytosolic sulfotransferases (SULTs): A systematic analysis.

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    Hui, Ying; Luo, Lijun; Zhang, Lingtian; Kurogi, Katsuhisa; Zhou, Chunyang; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh

    2015-07-01

    Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  8. SULT1A1 copy number variation: ethnic distribution analysis in an Indian population.

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    Almal, Suhani; Padh, Harish

    2017-11-01

    Cytosolic sulfotransferases (SULTs) are phase II detoxification enzymes involved in metabolism of numerous xenobiotics, drugs and endogenous compounds. Interindividual variation in sulfonation capacity is important for determining an individual's response to xenobiotics. SNPs in SULTs, mainly SULT1A1 have been associated with cancer risk and also with response to therapeutic agents. Copy number variation (CNVs) in SULT1A1 is found to be correlated with altered enzyme activity. This short report primarily focuses on CNV in SULT1A1 and its distribution among different ethnic populations around the globe. Frequency distribution of SULT1A1 copy number (CN) in 157 healthy Indian individuals was assessed using florescent-based quantitative PCR assay. A range of 1 to >4 copies, with a frequency of SULT1A1 CN =2 (64.9%) the highest, was observed in our (Indian) population. Upon comparative analysis of frequency distribution of SULT1A1 CN among diverse population groups, a statistically significant difference was observed between Indians (our data) and African-American (AA) (p = 0.0001) and South African (Tswana) (p populations. Distribution of CNV in the Indian population was found to be similar to that in European-derived populations of American and Japanese. CNV of SULT1A1 varies significantly among world populations and may be one of the determinants of health and diseases.

  9. Molecular characterization of novel sulfotransferases from the tick, Ixodes scapularis

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    King Roberta S

    2011-06-01

    Full Text Available Abstract Background Ixodes scapularis, commonly known as the blacklegged or deer tick, is the main vector of Lyme disease in the United States. Recent progress in transcriptome research has uncovered hundreds of different proteins expressed in the salivary glands of hard ticks, the majority of which have no known function, and include many novel protein families. We recently identified transcripts coding for two putative cytosolic sulfotransferases in these ticks which recognized phenolic monoamines as their substrates. In this current study, we characterize the genetic expression of these two cytosolic sulfotransferases throughout the tick life cycle as well as the enzymatic properties of the corresponding recombinant proteins. Interestingly, the resultant recombinant proteins showed sulfotransferase activity against both neurotransmitters dopamine and octopamine. Results The two sulfotransferase genes were coded as Ixosc SULT 1 & 2 and corresponding proteins were referred as Ixosc Sult 1 and 2. Using gene-specific primers, the sulfotransferase transcripts were detected throughout the blacklegged tick life cycle, including eggs, larvae, nymphs, adult salivary glands and adult midgut. Notably, the mRNA and protein levels were altered upon feeding during both the larval and nymphal life stages. Quantitative PCR results confirm that Ixosc SULT1 was statistically increased upon blood feeding while Ixosc SULT 2 was decreased. This altered expression led us to further characterize the function of these proteins in the Ixodid tick. The sulfotransferase genes were cloned and expressed in a bacterial expression system, and purified recombinant proteins Ixosc Sult 1(R and 2(R showed sulfotransferase activity against neurotransmitters dopamine and octopamine as well as the common sulfotransferase substrate p-nitrophenol. Thus, dopamine- or octopamine-sulfonation may be involved in altering the biological signal for salivary secretion in I. scapularis

  10. Farnesoid X receptor regulates SULT1E1 expression through inhibition of PGC1α binding to HNF4α.

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    Wang, Shuai; Yuan, Xue; Lu, Danyi; Guo, Lianxia; Wu, Baojian

    2017-12-01

    Sulfotransferase 1E1 (SULT1E1, also known as estrogen sulfotransferase) plays an important role in metabolism and detoxification of many endogenous and exogenous compounds (e.g., estrogens and flavonoids). Here we aimed to assess the effects of farnesoid X receptor (FXR) activation on SULT1E1 expression, and to determine the mechanism thereof. Treatment with specific FXR agonists (i.e., GW4064 and CDCA) significantly decreased both mRNA and protein levels of SULT1E1 in HepG2 cells. This was accompanied by a decrease in the enzymatic activity. The inhibitory effect was potentiated by FXR overexpression but attenuated by FXR knockdown, confirming FXR-dependent regulation of SULT1E1. Surprisingly, direct regulation of SULT1E1 by FXR was unlikely because FXR did not bind to SULT1E1 promoter or enhancer as revealed by chromatin immunoprecipitation (ChIP). Interestingly, SULT1E1 regulation was abolished when HNF4α (hepatocyte nuclear factor 4α, a known activator of SULT1E1) was silenced, supporting a critical role for HNF4α in FXR regulation of SULT1E1. Furthermore, a combination of ChIP, luciferase reporter and co-immunoprecipitation assays showed that FXR inhibited HNF4α transactivation of SULT1E1 by suppressed binding of the co-activator PGC1α (peroxisome proliferator-activated receptor-γ coactivator 1α) to HNF4α. In conclusion, FXR transcriptionally regulates SULT1E1 through inhibition of PGC1α binding to HNF4α. Targeting the FXR-SULT1E1 axis may represent a promising approach for management of estrogen-related diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Identification of Human Sulfotransferases Involved in Lorcaserin N-Sulfamate Formation.

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    Sadeque, Abu J M; Palamar, Safet; Usmani, Khawja A; Chen, Chuan; Cerny, Matthew A; Chen, Weichao G

    2016-04-01

    Lorcaserin [(R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine] hydrochloride hemihydrate, a selective serotonin 5-hydroxytryptamine (5-HT) 5-HT(2C) receptor agonist, is approved by the U.S. Food and Drug Administration for chronic weight management. Lorcaserin is primarily cleared by metabolism, which involves multiple enzyme systems with various metabolic pathways in humans. The major circulating metabolite is lorcaserin N-sulfamate. Both human liver and renal cytosols catalyze the formation of lorcaserin N-sulfamate, where the liver cytosol showed a higher catalytic efficiency than renal cytosol. Human sulfotransferases (SULTs) SULT1A1, SULT1A2, SULT1E1, and SULT2A1 are involved in the formation of lorcaserin N-sulfamate. The catalytic efficiency of these SULTs for lorcaserin N-sulfamate formation is widely variable, and among the SULT isoforms SULT1A1 was the most efficient. The order of intrinsic clearance for lorcaserin N-sulfamate is SULT1A1 > SULT2A1 > SULT1A2 > SULT1E1. Inhibitory effects of lorcaserin N-sulfamate on major human cytochrome P450 (P450) enzymes were not observed or minimal. Lorcaserin N-sulfamate binds to human plasma protein with high affinity (i.e., >99%). Thus, despite being the major circulating metabolite, the level of free lorcaserin N-sulfamate would be minimal at a lorcaserin therapeutic dose and unlikely be sufficient to cause drug-drug interactions. Considering its formation kinetic parameters, high plasma protein binding affinity, minimal P450 inhibition or induction potential, and stability, the potential for metabolic drug-drug interaction or toxicological effects of lorcaserin N-sulfamate is remote in a normal patient population. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  12. Gender difference in NASH susceptibility: Roles of hepatocyte Ikkβ and Sult1e1

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    Matsushita, Noriko; Hassanein, Mohamed T.; Martinez-Clemente, Marcos; Lazaro, Raul; French, Samuel W.; Xie, Wen; Lai, Keane; Karin, Michael; Tsukamoto, Hidekazu

    2017-01-01

    Myeloid cell and hepatocyte IKKβ may mediate the genesis of obesity and insulin resistance in mice fed high fat diet. However, their gender-specific roles in the pathogenesis of non-alcoholic steatohepatitis (NASH) are not known. Here we demonstrate myeloid IKKβ deficiency prevents Western diet-induced obesity and visceral adiposity in females but not in males, and attenuates hyperglycemia, global IR, and NASH in both genders. In contrast, all metabolic sequela including NASH are aggravated by hepatocyte IKKβ deficiency (IkbkbΔhep) in male but not female mice. Gene profiling identifies sulfotransferase family 1E (Sult1e1), which encodes a sulfotransferase E1 responsible for inactivation of estrogen, as a gene upregulated in NASH in both genders and most conspicuously in male IkbkbΔhep mice having worst NASH and lowest plasma estradiol levels. LXRα is enriched to LXRE on Sult1e1 promoter in male WT and IkbkbΔhep mice with NASH, and a Sult1e1 promoter activity is increased by LXRα and its ligand and augmented by expression of a S32A mutant of IκBα. These results demonstrate striking gender differences in regulation by IKKβ of high cholesterol saturated fat diet-induced metabolic changes including NASH and suggest hepatocyte IKKβ is protective in male due at least in part to its ability to repress LXR-induced Sult1e1. Our findings also raise a caution for systemic IKK inhibition for the treatment of NASH as it may exacerbate the disease in male patients. PMID:28797077

  13. Gender difference in NASH susceptibility: Roles of hepatocyte Ikkβ and Sult1e1.

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    Noriko Matsushita

    Full Text Available Myeloid cell and hepatocyte IKKβ may mediate the genesis of obesity and insulin resistance in mice fed high fat diet. However, their gender-specific roles in the pathogenesis of non-alcoholic steatohepatitis (NASH are not known. Here we demonstrate myeloid IKKβ deficiency prevents Western diet-induced obesity and visceral adiposity in females but not in males, and attenuates hyperglycemia, global IR, and NASH in both genders. In contrast, all metabolic sequela including NASH are aggravated by hepatocyte IKKβ deficiency (IkbkbΔhep in male but not female mice. Gene profiling identifies sulfotransferase family 1E (Sult1e1, which encodes a sulfotransferase E1 responsible for inactivation of estrogen, as a gene upregulated in NASH in both genders and most conspicuously in male IkbkbΔhep mice having worst NASH and lowest plasma estradiol levels. LXRα is enriched to LXRE on Sult1e1 promoter in male WT and IkbkbΔhep mice with NASH, and a Sult1e1 promoter activity is increased by LXRα and its ligand and augmented by expression of a S32A mutant of IκBα. These results demonstrate striking gender differences in regulation by IKKβ of high cholesterol saturated fat diet-induced metabolic changes including NASH and suggest hepatocyte IKKβ is protective in male due at least in part to its ability to repress LXR-induced Sult1e1. Our findings also raise a caution for systemic IKK inhibition for the treatment of NASH as it may exacerbate the disease in male patients.

  14. Human Sulfotransferases Enhance the Cytotoxicity of Tolvaptan.

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    Fang, Jia-Long; Wu, Yuanfeng; Gamboa da Costa, Gonçalo; Chen, Si; Chitranshi, Priyanka; Beland, Frederick A

    2016-03-01

    Tolvaptan, a vasopressin receptor 2 antagonist used to treat hyponatremia, has recently been reported to be associated with liver injury. Sulfotransferases (SULTs) have been implicated as important detoxifying and/or activating enzymes for numerous xenobiotics, drugs, and endogenous compounds. To characterize better the role of SULTs in tolvaptan metabolism, HEK293 cells stably overexpressing 12 human SULTs were generated. Using these cell lines, the extent of tolvaptan sulfate formation was assessed by reversed-phase high-performance liquid chromatography through comparison to a synthetic standard. Of the 12 known human SULTs, no detectable sulfation of tolvaptan was observed with SULT1A1, SULT1A2, SULT1A3, SULT1C2, SULT1C4, SULT4A1, or SULT6B1. The affinity of individual SULT isozymes, as determined by Km analysis, was SULT1C3 > SULT2A1 > SULT2B1 ∼ SULT1B1 > SULT1E1. The half inhibitory concentration of tolvaptan on cell growth in HEK293/SULT1C3 cells and HEK293/CYP3A4 & SULT1C3 cells was significantly lower than that in the corresponding HEK293/vector cells or HEK293/CYP3A4 & SULT vector cells. Moreover, exposing cells to tolvaptan in the presence of cyclosporine A, an inhibitor of the drug efflux transporters, significantly increased the intracellular levels of tolvaptan sulfate and decreased the cell viability in HEK293/SULT1C3 cells. These data indicate that sulfation increased the cytotoxicity of tolvaptan. Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.

  15. Methyleugenol DNA adducts in human liver are associated with SULT1A1 copy number variations and expression levels.

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    Tremmel, Roman; Herrmann, Kristin; Engst, Wolfram; Meinl, Walter; Klein, Kathrin; Glatt, Hansruedi; Zanger, Ulrich M

    2017-10-01

    Methyleugenol is a rodent hepatocarcinogen occurring in many herbs and spices as well as essential oils used for flavoring. Following metabolic activation by cytochromes P450 (CYPs) and sulfotransferases (SULTs), methyleugenol can form DNA adducts. Previously, we showed that DNA adduct formation by methyleugenol in mouse liver is dependent on SULT1A1 expression and that methyleugenol DNA adducts are abundant in human liver specimens. In humans, SULT1A1 activity is affected by genetic polymorphisms, including single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs). Here we investigated the relationship between individual methyleugenol DNA adduct levels and SULT1A1 in human liver samples. Using isotope-dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry, we quantified methyleugenol DNA adducts in 121 human surgical liver samples. Frequent CNVs, including deletions (f = 3.3%) and duplications (f = 36.4%) of SULT1A1, were identified using qPCR and TaqMan assays in the donors' genomic DNA. SULT1A1 mRNA and protein levels were quantified using microarray data and Western blot analysis, respectively. Methyleugenol DNA adducts were detected in all 121 liver samples studied. Their levels varied 122-fold between individuals and were significantly correlated to both mRNA and protein levels of SULT1A1 (r s = 0.43, and r s = 0.44, respectively). Univariate and multivariate statistical analysis identified significant associations of SULT1A1 CNVs with mRNA (p = 1.7 × 10-06) and protein (p = 4.4 × 10- 10) levels as well as methyleugenol DNA adduct levels (p = 0.003). These data establish the importance of SULT1A1 genotype for hepatic methyleugenol DNA adducts in humans, and they confirm a strong impact of SULT1A1 CNVs on SULT1A1 hepatic phenotype.

  16. Effects of SULT1A1 Copy Number Variation on Estrogen Concentration and Tamoxifen-Associated Adverse Drug Reactions in Premenopausal Thai Breast Cancer Patients: A Preliminary Study.

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    Charoenchokthavee, Wanaporn; Ayudhya, Duangchit Panomvana Na; Sriuranpong, Virote; Areepium, Nutthada

    2016-01-01

    Tamoxifen is a pharmacological estrogen inhibitor that binds to the estrogen receptor (ER) in breast cells. However, it shows an estrogenic effect in other organs, which causes adverse drug reactions (ADRs). The sulfotransferase 1A1 (SULT1A1) enzyme encoded by the SULT1A1 gene is involved in estrogen metabolism. Previous research has suggested that the SULT1A1 copy number is linked with the plasma estradiol (E2) concentration. Here, a total of 34 premenopausal breast cancer patients, selected from the Thai Tamoxifen (TTAM) Project, were screened for their SULT1A1 copy number, plasma E2 concentration and ADRs. The mean age was 44.3±11.1 years, and they were subtyped as ER+/ progesterone receptor (PR) + (28 patients), ER+/ PR- (5 patients) and ER-/PR- (1 patient). Three patients reported ADRs, which were irregular menstruation (2 patients) and vaginal discharge (1 patient). Most (33) patients had two SULT1A1 copies, with one patient having three copies. The median plasma E2 concentration was 1,575.6 (IQR 865.4) pg/ml. Patients with ADRs had significantly higher plasma E2 concentrations than those patients without ADRs (p = 0.014). The plasma E2 concentration was numerically higher in the patient with three SULT1A1 copies, but this lacked statistical significance.

  17. SULT1A3-Mediated Regiospecific 7-O-Sulfation of Flavonoids in Caco-2 Cells Can Be Explained by the Relevant Molecular Docking Studies

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    Meng, Shengnan; Wu, Baojian; Singh, Rashim; Yin, Taijun; Morrow, John Kenneth; Zhang, Shuxing; Hu, Ming

    2012-01-01

    Flavonoids are the polyphenolic compounds with various claimed health benefits, but the extensive metabolism by uridine-5'-diphospho-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) in liver and intestine led to poor oral bioavailabilities. The effects of structural changes on the sulfonation of flavonoids have not been systemically determined, although relevant effects of structural changes on the glucuronidation of flavonoids had. We performed the regiospecific sulfonation of sixteen flavonoids from five different subclasses of flavonoids, which are represented by apigenin (flavone), genistein (isoflavone), naringenin (flavanone), kaempherol (flavonol), and phloretin (chalcone). Additional studies were performed using 4 mono-hydroxyl flavonoids with –OH group at 3, 4’, 5 or 7 position, followed by 5 di-hydroxyl-flavonoids, and 2 tri-hydroxyl flavonoids by using expressed human SULT1A3 and Caco-2 cell lysates. We found that these compounds were exclusively sulfated at the 7-OH position by SULT1A3 and primarily sulfated at 7-OH position in Caco-2 cell lysates with minor amounts of 4’-O-sulfates formed as well. Sulfonation rates measured using SULT1A3 and Caco-2 cell lysates were highly correlated at substrate concentrations of 2.5 and 10 µM. Molecular docking studies provided structural explanations as to why sulfonation only occurred at the 7-OH position of flavones, flavonols and flavanones. In conclusion, molecular docking studies explain why SULT1A3 exclusively mediates sulfonation at the 7-OH position of flavones/flavonols, and correlation studies indicate that SULT1A3 is the main isoform responsible for flavonoid sulfonation in the Caco-2 cells. PMID:22352375

  18. Inhibition of human phenol and estrogen sulfotransferase by certain non-steroidal anti-inflammatory agents

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    King, Roberta S.; Ghosh, Anasuya A.; Wu, Jinfang

    2006-01-01

    This study was initiated on the hypothesis that aryl acetic acid and aryl carboxylic acid-containing drugs would inhibit human phenol sulfotransferase (SULT1A1), and that isoform selectivity would depend on the interaction of the aryl portion of the molecule with the acceptor binding site of the sulfotransferase. This hypothesis was based on results with the rat orthologue enzyme showing that oxidation of phenolic substrates to carboxylic acid derivatives resulted in competitive inhibition of...

  19. Action of Halowax 1051 on Enzymes of Phase I (CYP1A1 and Phase II (SULT1A and COMT Metabolism in the Pig Ovary

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    Justyna Barć

    2013-01-01

    Full Text Available Polychlorinated naphthalenes (PCNs are a group of organochlorinated compounds exhibiting dioxin-like properties. Previously published data showed the direct action of PCN-rich Halowax 1051 on ovarian follicular steroidogenesis. Taking into consideration that the observed biological effects of PCNs may be frequently side effects of metabolites generated by their detoxification, the aim of this study was to determine the activity and expression of enzymes involved in phase I (cytochrome P450, family 1 (CYP1A1 and phase II (sulfotransferase (SULT1A and catechol-O-methyltransferase (COMT detoxification metabolism. Cocultures of granulosa and theca interna cells collected from sexually mature pigs were exposed to 1 pg/mL to 10 ng/mL of Halowax 1051 for 1 to 48 hours, after which levels and activities of CYP1A1, SULT1A, and COMT were measured. Dose-dependent increases of CYP1A1 activity and expression were observed. High doses of Halowax 1051 were inhibitory to COMT and SULT1A activity and reduced their protein levels. In conclusion, fast activation of phase I enzymes with simultaneous inhibition of phase II enzymes indicates that the previously observed effect of Halowax 1051 on follicular steroidogenesis may partially result from metabolite action occurring locally in ovarian follicles.

  20. Combined effects of NQO1 Pro187Ser or SULT1A1 Arg213His polymorphism and smoking on bladder cancer risk: Two meta-analyses

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    Xiao-Chun Wang

    2017-10-01

    Full Text Available Objectives: Objectives: Cigarette smoking is the major risk factor of bladder cancer via exposure to chemical carcinogens. Nicotinamide adenine dinucleotide phosphate (NADP+: quinine oxidoreductase 1 (NQO1 and sulfotransferase 1A1 (SULT1A1 have been reported to involve in the metabolism of polycyclic aromatic hydrocarbons (PAHs and aromatic amines. Therefore, the risk of bladder cancer (BC may be influenced by polymorphisms in the genes that modulate metabolic detoxification in particular by interacting with cigarette smoking. Considering the limited power by the individual studies with a relatively small sample size, especially when analyzing the combined effect of polymorphisms in NQO1 and SULT1A1 genes and smoking, these 2 meta-analyses have aimed to clarify the combined effects of them on BC risk by integrating related studies. Material and Methods: Two meta-analyses included 1341 cases and 1346 controls concerning NQO1 Pro187Ser and smoking, and 1921 cases and 1882 controls on SULT1A1 Arg213His and smoking were performed. Odds ratios (OR and 95% confidence intervals (CI were used for assessing the strength of the association. Results: The result has demonstrated that smokers with NQO1 Pro/Ser or Ser/Ser genotypes have a prominent association with the risk of BC as compared with non-smokers with NQO1 Pro/Pro genotype, with OR equal to 3.71 (95% CI: 2.87–4.78, pheterogeneity = 0.376. Besides, smokers carrying SULT1A1 Arg/Arg genotypes were observed to confer 2.38 fold increased risk of BC (95% CI: 1.44–3.93, pheterogeneity = 0.001 when compared with non-smokers with SULT1A1 Arg/Arg or His/His genotypes. Conclusions: These findings have suggested that the NQO1 Pro187Ser or SULT1A1 Arg213His polymorphism combination with smoking significantly confer susceptibility to BC. Int J Occup Med Environ Health 2017;30(5:791–802

  1. Occurrence of sulfonated steroids and ovarian expression of steroid sulfatase and SULT1E1 in cyclic cows.

    Science.gov (United States)

    Blaschka, Carina; Schuler, Gerhard; Sánchez-Guijo, Alberto; Zimmer, Bettina; Feller, Sabine; Kotarski, Franziska; Wudy, Stefan A; Wrenzycki, Christine

    2017-12-17

    Historically sulfonated steroids were primarily considered as inactive metabolites destined for elimination. However, more recently they have been increasingly recognized as precursors for the production of bioactive steroids in target tissues and as functional molecules without preceding hydrolysis. In order to comprehensively characterize their occurrence in cyclic cows and their formation and hydrolysis in bovine ovarian steroidogenesis, ovaries from cyclic cows were screened for the expression of oestrogen sulfotransferase (SULTE1) and steroid sulfatase (STS) by Western blot and immunohistochemistry. Moreover, a broad spectrum of 13 sulfonated steroids was measured applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) in blood samples collected from three cycling heifers during defined stages of the ovarian cycle and in fluid obtained from ovarian follicles of different size. SULT1E1 was undetectable in ovarian tissues. For STS only a weak immunostaining was found predominantly in granulosa cells of larger follicles. However, no specific band occurred in Western blot. In blood, concentrations of all sulfonated steroids investigated were below the limit of quantification (LOQ). In follicular fluid, only cholesterol sulfate was measured in considerable concentrations (328.3 ± 63.8 ng/ml). However, the role of cholesterol sulfate in bovine follicular steroidogenesis remains unclear as concentrations were obviously unrelated to follicular size. The remaining sulfonated steroids investigated were undetectable or only slightly exceeded LOQ in a minor proportion of samples. The results are clearly contrary to a role of sulfonated steroids as important precursors, intermediates or products of bovine ovarian steroidogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Quantification of Sulfotransferases 1A1 and 1A3/4 in Tissue Fractions and Cell Lines by Multiple Reaction Monitoring Mass Spectrometry.

    Science.gov (United States)

    Yoshitake, Sho; McKay-Daily, Melissa; Tanaka, Masaki; Huang, Zeqi

    2017-11-17

    Within the sulfotransferase (SULT) superfamily of metabolic enzymes, SULT1A1 and 1A3/4 isoforms are of particular interest, due to their abilities to catalyze the sulfation of phenolic endobiotics and xenobiotics. Although the difference in their substrate specificity is well documented, an isoform-specific quantification method is still not available. To detect and quantify SULT1A1 and 1A3/4 in S9 fractions and cell lines using targeted mass spectrometry-based proteomics. Samples were tryptically digested, and signature peptides were quantified using liquid chromatography- multiple reaction monitoring mass spectrometry (LC-MRM/MS). Stable isotopelabeled (SIL) peptides were used as internal and calibration standards. SULT1A1 and SULT1A3/4 were quantified in various S9 fractions and cell line samples. Intraday and interday variabilities were low for relative quantification in S9 and cell line matrices (fractions and lentiviral transduced SULT1A-overexpressing cell lines. A reproducible method for simultaneous quantification of SULT1A1 and SULT1A3/4 in S9 fractions and cell line samples was established and validated. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. In silico mechanistic profiling to probe small molecule binding to sulfotransferases.

    Directory of Open Access Journals (Sweden)

    Virginie Y Martiny

    Full Text Available Drug metabolizing enzymes play a key role in the metabolism, elimination and detoxification of xenobiotics, drugs and endogenous molecules. While their principal role is to detoxify organisms by modifying compounds, such as pollutants or drugs, for a rapid excretion, in some cases they render their substrates more toxic thereby inducing severe side effects and adverse drug reactions, or their inhibition can lead to drug-drug interactions. We focus on sulfotransferases (SULTs, a family of phase II metabolizing enzymes, acting on a large number of drugs and hormones and showing important structural flexibility. Here we report a novel in silico structure-based approach to probe ligand binding to SULTs. We explored the flexibility of SULTs by molecular dynamics (MD simulations in order to identify the most suitable multiple receptor conformations for ligand binding prediction. Then, we employed structure-based docking-scoring approach to predict ligand binding and finally we combined the predicted interaction energies by using a QSAR methodology. The results showed that our protocol successfully prioritizes potent binders for the studied here SULT1 isoforms, and give new insights on specific molecular mechanisms for diverse ligands' binding related to their binding sites plasticity. Our best QSAR models, introducing predicted protein-ligand interaction energy by using docking, showed accuracy of 67.28%, 78.00% and 75.46%, for the isoforms SULT1A1, SULT1A3 and SULT1E1, respectively. To the best of our knowledge our protocol is the first in silico structure-based approach consisting of a protein-ligand interaction analysis at atomic level that considers both ligand and enzyme flexibility, along with a QSAR approach, to identify small molecules that can interact with II phase dug metabolizing enzymes.

  4. [A case-control study on association of SULT1A1 polymorphism, smoked meat intake with breast cancer risk].

    Science.gov (United States)

    Tao, Ping; Li, Hui; Wang, Qiong; Cao, Lan-qing; Li, Jia-yuan; Yang, Fei; Wang, Yuan-ping

    2012-09-01

    To assess the association of smoked meat intake, SULT1A1 polymorphism as well as their combined effects with breast cancer risk. A total of 400 newly diagnosed breast cancer cases from a cancer hospital in Sichuan province and 400 healthy controls from participants of physical examination in a hospital in Chengdu city were recruited from May 2007 to July 2009. A valid questionnaire was designed to collect their demographic characteristics and breast cancer risk factors. Daily intake of foods was collected using semi-quantitative frequency questionnaire and then the daily intake of smoked meat was calculated and transformed to energy-adjusted smoked meat intake by the residual method. Gene sequencing was used to analyze SULT1A1 Arg213His genotypes. Multivariable conditional logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (95%CIs). The energy-adjusted daily intake of smoked meat (Median (P₂₅, P₇₅)) was 8.65 (3.63, 18.44) g/d in cases and 4.44 (0.19, 8.71) g/d in controls. The frequency of SULT1A1 variant allele was 14.75% (59/400) among cases and 12.75% (51/400) among controls. High energy-adjusted daily intake of smoked meat (≥ 4.44 g/d) was significantly associated with breast cancer risk among premenopausal (OR = 2.31, 95%CI: 1.46 - 3.66) and postmenopausal subjects (OR = 3.13, 95%CI: 1.89 - 5.17). High energy-adjusted daily intake of smoked meat combined with carrying SULT1A1 variant allele elevated breast cancer risk among premenopausal (OR = 3.31, 95%CI: 1.66 - 6.62) and postmenopausal subjects (OR = 3.81, 95%CI: 1.79 - 8.10). High smoked meat intake contributes to high risk of breast cancer. SULT1A1 variant allele increases breast cancer risk among subjects who were exposed to high smoked meat intake.

  5. In vivo imaging of sulfotransferases

    Science.gov (United States)

    Barrio, Jorge R; Kepe, Vladimir; Small, Gary W; Satyamurthy, Nagichettiar

    2013-02-12

    Radiolabeled tracers for sulfotransferases (SULTs), their synthesis, and their use are provided. Included are substituted phenols, naphthols, coumarins, and flavones radiolabeled with .sup.18F, .sup.123I, .sup.124I, .sup.125I, or .sup.11C. Also provided are in vivo techniques for using these and other tracers as analytical and diagnostic tools to study sulfotransferase distribution and activity, in health and disease, and to evaluate therapeutic interventions.

  6. Modified Ames test using a strain expressing human sulfotransferase 1C2 to assess the mutagenicity of methyleugenol.

    Science.gov (United States)

    Honda, Hiroshi; Minegawa, Kazuyuki; Fujita, Yurika; Yamaguchi, Noriko; Oguma, Yoshihiro; Glatt, Hansruedi; Nishiyama, Naohiro; Kasamatsu, Toshio

    2016-01-01

    Several alkenylbenzenes, including methyleugenol (ME), are present in a wide range of botanicals and exhibit carcinogenic and mutagenic properties. Negative results are generally obtained for alkenylbenzenes in standard in vitro genotoxicity tests, including the Ames test. A lack of mutagenicity observed in such tests is thought to result from impaired metabolic activation of alkenylbenzenes via hydroxylation, with subsequent sulfoconjugation to its ultimate mutagenic or carcinogenic form. Although recent studies have reported the mutagenicity of hydroxylated ME metabolites in the Ames test using modified TA100 strains expressing human sulfotransferases (SULTs), to our knowledge, the detection of ME mutagenicity has not yet been reported. Using strain TA100-hSULT1C2, which expresses human SULT1C2, we optimized the protein content of S9 Mix and the pre-incubation time required to promote metabolic activation in the Ames test. This procedure enabled us to obtain a positive response with ME. We established Ames-test conditions enabling the detection of ME-induced mutagenicity, using a strain expressing human SULT1C2 in the presence of induced-rat S9 Mix. This simple approach will help assess the mutagenicity of other alkenylbenzenes and related chemicals.

  7. Intestinal carcinogenesis of two food processing contaminants, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 5-hydroxymethylfurfural, in transgenic FVB min mice expressing human sulfotransferases.

    Science.gov (United States)

    Svendsen, Camilla; Meinl, Walter; Glatt, Hansruedi; Alexander, Jan; Knutsen, Helle K; Hjertholm, Hege; Rasmussen, Tone; Husøy, Trine

    2012-12-01

    Humans express sulfotransferases (SULTs) of the SULT1A subfamily in many tissues, whilst the single SULT1A gene present in rodents is mainly expressed in liver. The food processing contaminants, 5-hydroxymethylfurfural (HMF) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are bioactivated by human SULT1A1 and SULT1A2. FVB multiple intestinal neoplasia (Min) mice, which spontaneously develop tumors and flat aberrant crypt foci (ACF) in intestine, were crossed with transgenic FVB mice expressing human SULT1A1 and 1A2 (hSULT) in several tissues, giving rise to wild-type and Min mice with and without hSULT. One-week-old Min mice with or without hSULT were given HMF (375 or 750 mg/kg bw) or saline by gavage three times a week for 11 wk. In another experiment, the F1 generation received subcutaneous injections of 50 mg/kg bw PhIP or saline 1 wk before birth, and 1, 2, and 3 wk after birth. HMF did not affect the formation of tumors, but may have induced some flat ACF (incidence 15-20%) in Min mice with and without hSULT. No control mouse developed any flat ACF. With the limitation that these putative effects were weak, they were unaffected by hSULT expression. The carcinogenic effect of PhIP increased in the presence of hSULT, with a significant increase in both incidence (31-80%) and number of colonic tumors (0.4-1.3 per animal). Thus, intestinal expression of human SULT1A1 and 1A2 might increase the susceptibility to compounds bioactivated via this pathway implying that humans might be more susceptible than conventional rodent models. Copyright © 2011 Wiley Periodicals, Inc.

  8. Combined effects of GSTO1 and SULT1A1 polymorphisms and cigarette smoking on urothelial carcinoma risk in a Taiwanese population

    Directory of Open Access Journals (Sweden)

    Min-Che Tung

    2014-09-01

    Conclusion: The present study provided epidemiological evidence for a significantly increased risk of UCB in ever smokers with the Ala/Ala genotype of the GSTO1 gene and the Arg/Arg genotype of the SULT1A1 gene.

  9. Role of human sulfotransferase 1A1 and N-acetyltransferase 2 in the metabolic activation of 16 heterocyclic amines and related heterocyclics to genotoxicants in recombinant V79 cells.

    Science.gov (United States)

    Chevereau, Matthieu; Glatt, Hansruedi; Zalko, Daniel; Cravedi, Jean-Pierre; Audebert, Marc

    2017-09-01

    Heterocyclic aromatic amines (HAAs) are primarily produced during the heating of meat or fish. HAAs are mutagenic and carcinogenic, and their toxicity in model systems depend on metabolic activation. This activation is mediated by cytochrome P450 (CYP) enzymes, in particular CYP1A2. Some studies have indicated a role of human sulfotransferase (SULT) 1A1 and N-acetyltransferase (NAT) 2 in the terminal activation of HAAs. In this study, we conducted a metabolism/genotoxicity relationship analysis for 16 HAAs and related heterocyclics. We used the γH2AX genotoxicity assay in V79 cells (deficient in CYP, SULT and NAT) and V79-derived cell lines genetically engineered to express human CYP1A2 alone or in combination with human SULT1A1 or NAT2. Our data demonstrated genotoxic properties for 13 out of the 16 compounds tested. A clear relationship between metabolic bioactivation and genotoxicity allowed to distinguish four groups: (1) Trp-P-1 genotoxicity was linked to CYP1A2 bioactivation only-with negligible effects of phase II enzymes; (2) Glu-P-2, Glu-P-1, Trp-P-2, APNH, MeAαC and AαC were bioactivated by CYP1A2 in combination with either phase II enzyme tested (NAT2 or SULT1A1); (3) IQ, 4-MeIQ, IQx, 8-MeIQx, and 4,8-DiMeIQx required CYP1A2 in combination with NAT2 to be genotoxic, whereas SULT1A1 did not enhance their genotoxicity; (4) PhIP became genotoxic after CYP1A2 and SULT1A1 bioactivation-NAT2 had not effect. Our results corroborate some previous data regarding the genotoxic potency of seven HAAs and established the genotoxicity mechanism for five others HAAs. This study also permits to compare efficiently the genotoxic potential of these 13 HAAs.

  10. North African genetic variation of cytochrome and sulfotransferase ...

    African Journals Online (AJOL)

    Sulfation pharmacogenetics: SULT1A1 and SULT1A2 allele frequencies in. Caucasian, Chinese and African-American subjects. Pharmacogenetics. 2001. Feb;11(1):57-68. Charles N. Falany,a, Dongning He,a Li Li,a Josie L. Falany,a Teresa W. Wilborn,b. Thomas A. Kocarek C and Runge-Morrisc M. Regulation of hepatic ...

  11. Sulfotransferase-1A1-dependent bioactivation of aristolochic acid I and N-hydroxyaristolactam I in human cells.

    Science.gov (United States)

    Hashimoto, Keiji; Zaitseva, Irina N; Bonala, Radha; Attaluri, Sivaprasad; Ozga, Katherine; Iden, Charles R; Johnson, Francis; Moriya, Masaaki; Grollman, Arthur P; Sidorenko, Viktoriya S

    2016-07-01

    Aristolochic acids (AA) are implicated in the development of chronic renal disease and upper urinary tract carcinoma in humans. Using in vitro approaches, we demonstrated that N-hydroxyaristolactams, metabolites derived from partial nitroreduction of AA, require sulfotransferase (SULT)-catalyzed conjugation with a sulfonyl group to form aristolactam-DNA adducts. Following up on this observation, bioactivation of AA-I and N-hydroxyaristolactam I (AL-I-NOH) was studied in human kidney (HK-2) and skin fibroblast (GM00637) cell lines. Pentachlorophenol, a known SULT inhibitor, significantly reduced cell death and aristolactam-DNA adduct levels in HK-2 cells following exposure to AA-I and AL-I-NOH, suggesting a role for Phase II metabolism in AA activation. A gene knockdown, siRNA approach was employed to establish the involvement of selected SULTs and nitroreductases in AA-I bioactivation. Silencing of SULT1A1 and PAPSS2 led to a significant decrease in aristolactam-DNA levels in both cell lines following exposure to AA-I, indicating the critical role for sulfonation in the activation of AA-I in vivo Since HK-2 cells proved relatively resistant to knockdown with siRNAs, gene silencing of xanthine oxidoreductase, cytochrome P450 oxidoreductase and NADPH:quinone oxidoreductase was conducted in GM00637 cells, showing a significant increase, decrease and no effect on aristolactam-DNA levels, respectively. In GM00637 cells exposed to AL-I-NOH, suppressing the SULT pathway led to a significant decrease in aristolactam-DNA formation, mirroring data obtained for AA-I. We conclude from these studies that SULT1A1 is involved in the bioactivation of AA-I through the sulfonation of AL-I-NOH, contributing significantly to the toxicities of AA observed in vivo. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Crystal Structure of StaL, A Glycopeptide Antibiotic Sulfotransferase from Streptomyces Toyocaensis

    Energy Technology Data Exchange (ETDEWEB)

    Shi,R.; Lamb, S.; Bhat, S.; Sulea, T.; Wright, G.; Matte, A.; Cygler, M.

    2007-01-01

    Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide antibiotics such as vanco-mycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934 [GenBank], unique teicoplanin-class glycopeptide antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 [GenBank] biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3'-phosphoadenosine 5'-phosphate. This is only the second prokaryotic sulfotransferase to be structurally characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys{sup 12}, His{sup 67}, and Ser{sup 98}), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate binding and/or activity, including Trp{sup 34}, His{sup 43}, Phe{sup 77}, Trp{sup 132}, and Glu{sup 205}.

  13. Biotransformation enzyme-dependent formation of micronucleus and multinuclei in cell line V79-hCYP2E1-hSULT1A1 by 2-nitropropane and N-nitrosodimethylamine.

    Science.gov (United States)

    Deng, Hong; Gao, Hai; Liu, Yungang

    2011-11-27

    V79-hCYP2E1-hSULT1A1, a V79-derived cell line co-expressing both human CYP2E1 and SULT1A1, has been constructed and efficiently used in detection of the mutagenic activities of a number of promutagens. 2-Nitropropane (2-NP) and N-nitrosodimethylamine (NDMA), both being hepatocarcinogenic to animals but inactive in standard genotoxicity assays in vitro, are activated to mutagenic metabolites by human SULT1A1 and CYP2E1, respectively. Nevertheless, little is known about the chromosomal effects of these two carcinogens. In the present study, we investigated the effects of 2-NP and NDMA on frequencies of micronucleated (F(mi)) and multinucleated cells (F(mu)) in V79-hCYP2E1-hSULT1A1 cells. The results showed induction of both F(mi) and F(mu) by 2-NP and NDMA individually, and this effect was completely suppressed by relatively specific inhibitor of SULT1A1 and CYP2E1, i.e., pentachlorophenol and 1-aminobenzotriazole, respectively. The F(mu)/F(mi) ratio in 2-NP groups was significantly higher than NDMA groups, probably indicating an aneugenic activity of 2-NP based on proposed F(mu)/F(mi) ratio as a simple index to discriminate aneugens from clastogens. The present study has established biotransformation enzyme-dependent formation of multinuclei and micronuclei induced by 2-NP and NDMA. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Characterization of iodothyronine sulfotransferase activity in rat liver

    NARCIS (Netherlands)

    E. Kaptein (Ellen); G.A.C. van Haasteren (Goedele); E. Linkels; W.J. de Greef; T.J. Visser (Theo)

    1997-01-01

    textabstractSulfation is an important pathway in the metabolism of thyroid hormone because it strongly facilitates the degradation of the hormone by the type I iodothyronine deiodinase. However, little is known about the properties and possible regulation of the sulfotransferase(s)

  15. Regulation of Sulfotransferase and UDP-Glucuronosyltransferase Gene Expression by the PPARs

    Directory of Open Access Journals (Sweden)

    Melissa Runge-Morris

    2009-01-01

    Full Text Available During phase II metabolism, a substrate is rendered more hydrophilic through the covalent attachment of an endogenous molecule. The cytosolic sulfotransferase (SULT and UDP-glucuronosyltransferase (UGT families of enzymes account for the majority of phase II metabolism in humans and animals. In general, phase II metabolism is considered to be a detoxication process, as sulfate and glucuronide conjugates are more amenable to excretion and elimination than are the parent substrates. However, certain products of phase II metabolism (e.g., unstable sulfate conjugates are genotoxic. Members of the nuclear receptor superfamily are particularly important regulators of SULT and UGT gene transcription. In metabolically active tissues, increasing evidence supports a major role for lipid-sensing transcription factors, such as peroxisome proliferator-activated receptors (PPARs, in the regulation of rodent and human SULT and UGT gene expression. This review summarizes current information regarding the regulation of these two major classes of phase II metabolizing enzyme by PPARs.

  16. Characterization of kidney sulfotransferases during lead-induced nephrotoxicity in rats

    Energy Technology Data Exchange (ETDEWEB)

    Templer, L.A.; Kong, J.; Ronis, M.J.J.; Ringer, D.P. [Univ. Arkansas Medical School, Little Rock, AR (United States)

    1996-03-08

    Kidney sulfotransferases (ST) have been shown to be involved in the biotransformation of steroid and thyroid hormones as well as xenobiotics varying from carcinogenic heterocyclic amines to drugs such as acetaminophen. In order to examine the impact of lead-induced nephrotoxicity on kidney aryl, estrogen and DHEA STs during growth and development, time-impregnated female Sprague-Dawley rats were exposed ad libitum to lead acetate (0.6%) in drinking water from gestational day 5 and continuing in male and female pups until they were sacrificed at day 85. Cytosols from male rat kidneys showed levels of estrogen ST activity (59% of females) that were significantly lowered (P{le}0.05) after lead exposure (6-20% of male). Aryl ST activity was relatively unchanged in male rats after rat kidney cytosol. Immunochemical analysis of cytosols from normal males and females with the antiserums to the three STs substantiated the presence of only the aryl and estrogen STs. Immunohistochemical techniques localized the aryl and estrogen STs primarily to the S3 section of the proximal tubules. These findings indicate that kidney STs may be differently modulated during lead exposure.

  17. On the Molecular Basis Underlying the Metabolism of Tapentadol Through Sulfation.

    Science.gov (United States)

    Bairam, Ahsan F; Rasool, Mohammed I; Kurogi, Katsuhisa; Liu, Ming-Cheh

    2017-01-10

    Previous studies reported that tapentadol-sulfate represented one of the major metabolites of tapentadol excreted in urine. The current study aimed to identify the human cytosolic sulfotransferases (SULTs) that is(are) capable of sulfating tapentadol and to examine whether human cells and human organ specimens are capable of sulfating tapentadol. Thirteen human SULTs, previously expressed and purified, as well as human organ cytosols, were analyzed for tapentadol-sulfating activity using an established sulfotransferase assay. Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [(35)S]sulfate in the presence of different concentrations of tapentadol. Three of the thirteen human SULTs, SULT1A1, SULT1A3, and SULT1C4, were found to display sulfating activity toward tapentadol. Kinetic analysis revealed that SULT1A3 displayed the highest catalytic efficiency in mediating the sulfation of tapentadol, followed by SULT1A1 and SULT1C4. Using cultured HepG2 and Caco-2 cells, the generation and release of sulfated tapentadol under metabolic conditions was demonstrated. Moreover, of the four human organ specimens (kidney, liver, lung, and small intestine) tested, the cytosols prepared from small intestine and liver showed significant tapentadol-sulfating capacity (at 0.0203 and 0.0054 nmol/min/mg, respectively). Taken together, the results derived from the current study provided a molecular basis underlying the sulfation of tapentadol in humans.

  18. Ethanol and 4-methylpyrazole increase DNA adduct formation of furfuryl alcohol in FVB/N wild-type mice and in mice expressing human sulfotransferases 1A1/1A2.

    Science.gov (United States)

    Sachse, Benjamin; Meinl, Walter; Glatt, Hansruedi; Monien, Bernhard H

    2016-03-01

    Furfuryl alcohol (FFA) is a carcinogenic food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. The activation by sulfotransferases (SULTs) yields a DNA reactive and mutagenic sulfate ester. The most prominent DNA adduct, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MF-dG), was detected in FFA-treated mice and also in human tissue samples. The dominant pathway of FFA detoxification is the oxidation via alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The activity of these enzymes may be greatly altered in the presence of inhibitors or competitive substrates. Here, we investigated the impact of ethanol and the ADH inhibitor 4-methylpyrazole (4MP) on the DNA adduct formation by FFA in wild-type and in humanized mice that were transgenic for human SULT1A1/1A2 and deficient in the mouse (m) Sult1a1 and Sult1d1 genes (h1A1/1A2/1a1(-)/1d1(-)). The administration of FFA alone led to hepatic adduct levels of 4.5 N(2)-MF-dG/10(8) nucleosides and 33.6 N(2)-MF-dG/10(8) nucleosides in male and female wild-type mice, respectively, and of 19.6 N(2)-MF-dG/10(8) nucleosides and 95.4 N(2)-MF-dG/10(8) nucleosides in male and female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 1.6g ethanol/kg body weight increased N(2)-MF-dG levels by 2.3-fold in male and by 1.7-fold in female wild-type mice and by 2.5-fold in male and by 1.5-fold in female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 100mg 4MP/kg body weight had a similar effect on the adduct levels. These findings indicate that modulators of the oxidative metabolism, e.g. the drug 4MP or consumption of alcoholic beverages, may increase the genotoxic effects of FFA also in humans. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Activation of the liver carcinogen 2-nitropropane by aryl sulfotransferase.

    Science.gov (United States)

    Sodum, R S; Sohn, O S; Nie, G; Fiala, E S

    1994-01-01

    8-Aminoguanine had previously been identified as one of the nucleic acid base modifications produced in livers of rats by treatment with the hepatocarcinogen 2-nitropropane (2-NP), and a hypothetical mechanism of activation of 2-NP to hydroxylamine-O-sulfonate or acetate that would lead to NH2+, an aminating species, was proposed [Sodum et al. (1993) Chem. Res. Toxicol. 6, 269-276]. We now present in vivo and in vitro experimental evidence for the activation of 2-NP to an aminating species by rat liver aryl sulfotransferase. Pretreatment of rats with the aryl sulfotransferase inhibitors pentachlorophenol or 2,6-dichloro-4-nitrophenol significantly decreased the levels of liver nucleic acid modifications produced by 2-NP treatment. Furthermore, partially purified rat liver aryl sulfotransferase was shown to activate 2-NP and 2-NP nitronate in vitro at neutral pH and 37 degrees C, to a reactive species that aminated guanosine at the C8 position. This activation was dependent on the presence of the enzyme, its specific cofactor adenosine 3'-phosphate 5'-phosphosulfate, and mercaptoethanol. As in the case of the in vitro studies, pentachlorophenol and 2,6-dichloro-4-nitrophenol inhibited the in vitro formation of 8-aminoguanosine and 8-oxoguanosine. The corresponding primary nitroalkane, 1-nitropropane, which is not mutagenic and does not appear to be carcinogenic, was not a substrate for aryl sulfotransferase in the in vitro amination of guanosine.

  20. Heparan sulfate 6-O-sulfotransferase is essential for muscle development in zebrafish

    OpenAIRE

    Bink, R.J.; Habuchi, H; Lele, Z.; Dolk, E.; Joore, J.; Rauch, G.; Geisler, R.; Wilson, S.W.; Hertog, J. den; Kimata, K.; Zivkovic, D.

    2003-01-01

    Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein show...

  1. Mechanisms of gender-specific regulation of mouse sulfotransferases (Sults)

    OpenAIRE

    Alnouti, Yazen; Klaassen, Curtis D.

    2010-01-01

    Marked gender differences in the expression of sulfotransferases (Sults) are known to exist in several species including rats, mice and hamsters. However, the mechanism for this gender difference is not known. Therefore, in the present study, it was determined whether sex and/or growth hormone (GH) are responsible for the gender difference in the expression of Sults using gonadectomized (GNX), hypophysectomized (HX) and GH-releasing hormone receptor-deficient little (lit/lit) mouse models.Sul...

  2. Sulfonation of raloxifene in HEK293 cells overexpressing SULT1A3: Involvement of breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 4 (MRP4/ABCC4) in excretion of sulfate metabolites.

    Science.gov (United States)

    Zhou, Xiaotong; Wang, Shaoxiang; Sun, Hua; Wu, Baojian

    2015-12-01

    Excretion of sulfate metabolites is an essential process in disposition of raloxifene via the sulfonation pathway. However, the transporters responsible for excretion of raloxifene sulfates remain undefined. Here, sulfonation of raloxifene and excretion of its sulfate metabolites were investigated using SULT1A3-overexpressing HEK293 cells (or SULT293 cells) with significant expression of BCRP and MRP4. SULT293 cell lysate catalyzed the sulfonation of raloxifene at both 6-OH and 4'-OH groups, generating raloxifene-6-sulfate (R-6-S) and raloxifene-4'-sulfate (R-4'-S), respectively. Sulfate formation followed the Michaelis-Menten kinetics (Km = 0.49 μM and Vmax = 5.79 pmol/min/mg for R-6-S; Km = 0.33 μM and Vmax = 1.25 pmol/min/mg for R-4'-S). As expected, the recombinant SULT1A3 enzyme showed a high similarity in raloxifene sulfonation profiles with the lysate preparation. Ko143, a selective inhibitor of BCRP, significantly decreased the excretion rates of raloxifene sulfates (maximal 66.1%) while increasing the intracellular sulfates (maximal 282%). As a result, the apparent efflux clearance (CLef,app, representing the efflux efficiency of raloxifene sulfates) was substantially reduced (maximal 85.6%). Likewise, the pan-MRP inhibitor MK-571 significantly deceased the excretion rates (maximal 69.6%) and CLef,app values (maximal 96.0%) of raloxifene sulfates while increasing the intracellular sulfates (maximal 667%). Further, the short-hairpin RNA (shRNA) targeting BCRP significantly reduced (maximal 35.0%) sulfate excretion. Use of BCRP shRNA also caused significant decreases (maximal 52.5%) in the CLef,app values. Silencing of MRP4 by shRNA led to a substantial alteration in sulfate disposition (i.e., 28.6-37.8% reductions in sulfate excretion, 30.5-59.3% elevations in intracellular sulfates, and 44.8-47.7% deceases in CLef,app values). In conclusion, two sulfate metabolites R-6-S and R-4'-S were generated from raloxifene in SULT293 cells. Cellular

  3. Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay.

    Science.gov (United States)

    Høie, Anja Hortemo; Svendsen, Camilla; Brunborg, Gunnar; Glatt, Hansruedi; Alexander, Jan; Meinl, Walter; Husøy, Trine

    2015-10-01

    The food processing contaminants 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 5-hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N-hydroxy-PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild-type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N-hydroxy-PhIP and HMF in vivo. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

  4. Cytosolic glutamine synthetase

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie; Eriksson, Ulf Dennis; Møller, Inge Skrumsager

    2014-01-01

    Overexpression of the cytosolic enzyme glutamine synthetase 1 (GS1) has been investigated in numerous cases with the goal of improving crop nitrogen use efficiency. However, the outcome has generally been inconsistent. Here, we review possible reasons underlying the lack of success and conclude...

  5. Liver X receptor alpha mediated genistein induction of human dehydroepiandrosterone sulfotransferase (hSULT2A1) in Hep G2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yue; Zhang, Shunfen [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078 (United States); Zhou, Tianyan [Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100083 (China); Huang, Chaoqun; McLaughlin, Alicia [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078 (United States); Chen, Guangping, E-mail: guangping.chen@okstate.edu [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078 (United States)

    2013-04-15

    Cytosolic sulfotransferases are one of the major families of phase II drug metabolizing enzymes. Sulfotransferase-catalyzed sulfonation regulates hormone activities, metabolizes drugs, detoxifies xenobiotics, and bioactivates carcinogens. Human dehydroepiandrosterone sulfotransferase (hSULT2A1) plays important biological roles by sulfating endogenous hydroxysteroids and exogenous xenobiotics. Genistein, mainly existing in soy food products, is a naturally occurring phytoestrogen with both chemopreventive and chemotherapeutic potential. Our previous studies have shown that genistein significantly induces hSULT2A1 in Hep G2 and Caco-2 cells. In this study, we investigated the roles of liver X receptor (LXRα) in the genistein induction of hSULT2A1. LXRs have been shown to induce expression of mouse Sult2a9 and hSULT2A1 gene. Our results demonstrate that LXRα mediates the genistein induction of hSULT2A1, supported by Western blot analysis results, hSULT2A1 promoter driven luciferase reporter gene assay results, and mRNA interference results. Chromatin immunoprecipitation (ChIP) assay results demonstrate that genistein increase the recruitment of hLXRα binding to the hSULT2A1 promoter. These results suggest that hLXRα plays an important role in the hSULT2A1 gene regulation. The biological functions of phytoestrogens may partially relate to their induction activity toward hydroxysteroid SULT. - Highlights: ► Liver X receptor α mediated genistein induction of hSULT2A1 in Hep G2 cells. ► LXRα and RXRα dimerization further activated this induction. ► Western blot results agreed well with luciferase reporter gene assay results. ► LXRs gene silencing significantly decreased hSULT2A1 expression. ► ChIP analysis suggested that genistein enhances hLXRα binding to the hSULT2A1 promoter.

  6. Tyrosylprotein sulfotransferase regulates collagen secretion in Caenorhabditis elegans.

    Science.gov (United States)

    Kim, Tai Hoon; Kim, Do Hyun; Nam, Hyung Wook; Park, Sang Yoon; Shim, Jaegal; Cho, Jin Won

    2010-04-01

    The sulfation of tyrosine residues is an important post-translational modification involved in the regulation of protein function. We examined the activity of worm tyrosylprotein sulfotransferase (TPST-1) on a typical cuticle collagen, ROL-6, in C. elegans. We verified that TPST-1 sulfates three tyrosine residues of ROL-6 in vitro. We found that these tyrosine residues are important for the secretion of ROL-6::GFP. Mutant ROL-6::GFP proteins that contain more than two substitutions of the target tyrosine residues are severely deficient in cuticle localization. Consistently, knock down of tpst-1 blocked the cuticle localization of ROL-6::GFP. Therefore, the sulfation of ROL-6 by TPST-1 is critical for the proper localization of ROL-6. We also confirmed that worm TPST-1 is localized to the trans-Golgi network (TGN). Our results indicate that TPST-1 regulates cuticle organization by promoting the transport of ROL-6 from the TGN to the cuticle.

  7. Silencing of Carbohydrate Sulfotransferase 15 Hinders Murine Pulmonary Fibrosis Development

    Directory of Open Access Journals (Sweden)

    Yoshiro Kai

    2017-03-01

    Full Text Available Pulmonary fibrosis is a progressive lung disorder characterized by interstitial fibrosis, for which no effective treatments are available. Chondroitin sulfate proteoglycan (CSPG has been shown to be a mediator, but the specific component of glycosaminoglycan chains of CSPG has not been explored. We show that chondroitin sulfate E-type (CS-E is involved in fibrogenesis. Small interfering RNA (siRNA targeting carbohydrate sulfotransferase 15 (CHST15 was designed to inhibit CHST15 mRNA and its product, CS-E. CS-E augments cell contraction and CHST15 siRNA inhibits collagen production. We found that bleomycin treatment increased CHST15 expression in interstitial fibroblasts at day 14. CHST15 siRNA was injected intranasally on days 1, 4, 8, and 11, and CHST15 mRNA was significantly suppressed by day 14. CHST15 siRNA reduced lung CSPG and the grade of fibrosis. CHST15 siRNA repressed the activation of fibroblasts, as evidenced by suppressed expression of α smooth muscle actin (αSMA, connective tissue growth factor (CTGF, lysyl oxidase like 2 (LOXL2, and CC-chemokine ligand 2 (CCL2/monocyte chemoattractant protein-1 (MCP-1. Inflammatory infiltrates in the bronchoalveolar lavage fluid (BALF and interstitium were diminished by CHST15 siRNA. These results indicate a pivotal role for CHST15 in fibroblast-mediated lung fibrosis and suggest a possible new therapeutic role for CHST15 siRNA in pulmonary fibrosis.

  8. Dissolving Lignin in Water through Enzymatic Sulfation with Aryl Sulfotransferase.

    Science.gov (United States)

    Prinsen, Pepijn; Narani, Anand; Hartog, Aloysius F; Wever, Ron; Rothenberg, Gadi

    2017-05-22

    We introduce the concept of using site-specific sulfation of various lignins for increasing their aqueous solubility and thereby their processability. Using p-nitrophenylsulfate as a sulfate source and an aryl sulfotransferase enzyme as catalyst, lignins are easily sulfated at ambient conditions. We demonstrate the specific sulfation of phenolic hydroxyl groups on five different lignins: Indulin AT (Kraft softwood), Protobind 1000 (mixed wheat straw/Sarkanda grass soda) and three organosolv lignins. The reaction proceeds smoothly and the increase in solubility is visible to the naked eye. We then examine the reaction kinetics, and show that these are easily monitored qualitatively and quantitatively using UV/Vis spectroscopy. The UV/Vis results are validated with (31) P NMR spectroscopy of the lignin phenol groups after derivatization with phosphorylation reagent II. In general, the results are more significant with organosolv lignins, as Kraft and soda lignins are produced from aqueous lignocellulose extraction processes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The 2.7 Å resolution structure of the glycopeptide sulfotransferase Teg14

    Energy Technology Data Exchange (ETDEWEB)

    Bick, Matthew J. [Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Banik, Jacob J. [Howard Hughes Medical Institute, Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Darst, Seth A. [Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Brady, Sean F., E-mail: sbrady@rockefeller.edu [Howard Hughes Medical Institute, Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States)

    2010-12-01

    The 2.7 Å resolution crystal structure of Teg14, a glycopeptide sulfotransferase cloned from an uncultured soil bacterium, is described. The relationship of Teg14 to other sulfotransferases is discussed. The TEG gene cluster was recently isolated from an environmental DNA library and is predicted to encode the biosynthesis of a polysulfated glycopeptide congener. Three closely related sulfotransferases found in the TEG gene cluster (Teg12, Teg13 and Teg14) have been shown to sulfate the teicoplanin aglycone at three unique sites. Crystal structures of the first sulfotransferase from the TEG cluster, Teg12, in complex with the teicoplanin aglycone and its desulfated cosubstrate PAP have recently been reported [Bick et al. (2010 ▶), Biochemistry, 49, 4159–4168]. Here, the 2.7 Å resolution crystal structure of the apo form of Teg14 is reported. Teg14 sulfates the hydroxyphenylglycine at position 4 in the teicoplanin aglycone. The Teg14 structure is discussed and is compared with those of other bacterial 3′-phosphoadenosine 5′-phosphosulfate-dependent sulfotransferases facilitating crystallographic experiments, especially in the field of microcrystallography.

  10. Cytosolic glutamine synthetase in barley

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie

    fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...

  11. Cytosolic delivery: Just passing through

    Science.gov (United States)

    Sánchez-Navarro, Macarena; Teixidó, Meritxell; Giralt, Ernest

    2017-08-01

    Intracellular protein delivery has been a major challenge in the field of cell biology for decades. Engineering such delivery is a key step in the development of protein- and antibody-based therapeutics. Now, two different approaches that enable the delivery of antibodies and antibody fragments into the cytosol have been developed.

  12. Heparan sulfate 6-O-Sulfotransferase is essential for muscle development in zebrafish

    NARCIS (Netherlands)

    Bink, R.J.; Habuchi, H.; Lele, Z.; Dolk, E.; Joore, J.; Rauch, G.; Geisler, R.; Wilson, S.W.; Hertog, J. den; Kimata, K.; Zivkovic, D.

    2003-01-01

    Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene

  13. The important roles of steroid sulfatase and sulfotransferases in gynecological diseases

    Directory of Open Access Journals (Sweden)

    Tea eLanisnik Rizner

    2016-02-01

    Full Text Available Gynecological diseases such as endometriosis, adenomyosis and uterine fibroids, and gynecological cancers including endometrial cancer and ovarian cancer, affect a large proportion of women. These diseases are estrogen dependent, and their progression often depends on local estrogen formation. In peripheral tissues, estrogens can be formed from the inactive precursors dehydroepiandrosterone sulfate and estrone sulfate. Sulfatase and sulfotransferases have pivotal roles in these processes, where sulfatase hydrolyzes estrone sulfate to estrone, and dehydroepiandrosterone sulfate to dehydroepiandrosterone, and sulfotransferases catalyze the reverse reactions. Further activation of estrone to the most potent estrogen, estradiol, is catalyzed by 17-ketosteroid reductases, while estradiol can be formed from dehydroepiandrosterone by the sequential actions of 3-hydroxysteroid dehydrogenase-4-isomerase, aromatase, and 17-ketosteroid reductase. This review introduces the sulfatase and sulfotransferase enzymes, in terms of their structures and reaction mechanisms, and the regulation and different transcripts of their genes, together with the importance of their currently known single nucleotide polymorphisms. Data on expression of sulfatase and sulfotransferases in gynecological diseases are also reviewed. There are often unchanged mRNA and protein levels in diseased tissue, with higher sulfatase activities in cancerous endometrium, ovarian cancer cell lines, and adenomyosis. This can be indicative of a disturbed balance between the sulfatase and sulfotransferases enzymes, defining the potential for sulfatase as a drug target for treatment of gynecological diseases. Finally, clinical trials with sulfatase inhibitors are discussed, where two inhibitors have already concluded phase II trials, although so far with no convincing clinical outcomes for patients with endometrial cancer and endometriosis.

  14. Sulfonation of 17{beta}-estradiol and inhibition of sulfotransferase activity by polychlorobiphenylols and celecoxib in channel catfish, Ictalurus punctatus

    Energy Technology Data Exchange (ETDEWEB)

    Wang Liquan [Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610 (United States); James, Margaret O. [Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610 (United States)]. E-mail: mojames@ufl.edu

    2007-03-10

    The sulfonation of 17{beta}-estradiol (E2) by human liver and recombinant sulfotransferases is influenced by environmental contaminants such as hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs), which are potent inhibitors, and the therapeutic drug, celecoxib, which affects positional sulfonation of E2. In some locations, the aquatic environment is contaminated by PCBs, OH-PCBs and widely used therapeutic drugs. The objectives of this study were to investigate the sulfonation kinetics of E2 in liver cytosol from channel catfish (Ictalurus punctatus); to examine the effect of OH-PCBs on E2 sulfonation; and to determine if celecoxib altered the position of E2 sulfonation, as it does with human liver cytosol. E2 was converted to both 3- and 17-sulfates by catfish liver cytosol. At E2 concentrations below 1{mu}M, formation of E2-3-sulfate (E2-3-S) predominated, but substrate inhibition was observed at higher concentrations. Rates of E2-3-S formation at different E2 concentrations were fit to a substrate inhibition model, with K{sup '}{sub m} and V{sup '}{sub max} values of 0.40+/-0.10{mu}M and 91.0+/-4.7pmol/min/mg protein, respectively and K{sub i} of 1.08+/-0.09{mu}M. The formation of E2-17-S fit Michaelis-Menten kinetics over the concentration range 25nM to 2.5{mu}M, with K{sub m} and V{sub max} values of 1.07+/-0.23{mu}M and 25.7+/-4.43pmol/min/mg protein, respectively. The efficiency (V{sub max}/K{sub m}) of formation of E2-3-S was 9.8-fold higher than that of E2-17-S. Several OH-PCBs inhibited E2 3-sulfonation, measured at an E2 concentration of 1nM. Of those tested, the most potent inhibitor was 4'-OH-CB79, with two chlorine atoms flanking the OH group (IC{sub 50}: 94nM). The inhibition of estrogen sulfonation by OH-PCBs may disrupt the endocrine system and thus contribute to the known toxic effects of these compounds. Celecoxib did not stimulate E2-17-S formation, as is the case with human liver cytosol, but did inhibit the

  15. Crystal Structures of the Glycopeptide Sulfotransferase Teg12 Complexed with the Teicoplanin Aglycone

    Science.gov (United States)

    Bick, Matthew J.; Banik, Jacob J.; Darst, Seth A.; Brady, Sean F.

    2010-01-01

    The TEG gene cluster, a glycopeptide biosynthetic gene cluster that is predicted to encode the biosynthesis of a polysulfated glycopeptide congener, was recently cloned from DNA extracted directly from desert soil. This predicted glycopeptide gene cluster contains three closely related sulfotransferases (Teg12, 13, and 14) that sulfate teicoplanin-like glycopeptides at three unique sites. Here we report a series of structures including: an apo structure of Teg12, Teg12 bound to the desulfated co-substrate 3'-phosphoadenosine 5'-phosphate and Teg12 bound to the teicoplanin aglycone. Teg12 appears to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding and catalysis. Loop regions that exhibit the most conformational flexibility show the least sequence conservation between TEG sulfotransferases. Site directed mutagenesis guided by our structural studies confirmed the importance of key catalytic residues as well as the importance of residues found throughout the conformationally flexible loop regions. PMID:20361791

  16. Crystal Structures of the Glycopeptide Sulfotransferase Teg12 in a Complex with the Teicoplanin Aglycone

    Energy Technology Data Exchange (ETDEWEB)

    Bick, Matthew J.; Banik, Jacob J.; Darst, Seth A.; Brady, Sean F. (Rockefeller)

    2010-06-25

    The TEG gene cluster, a glycopeptide biosynthetic gene cluster that is predicted to encode the biosynthesis of a polysulfated glycopeptide congener, was recently cloned from DNA extracted directly from desert soil. This predicted glycopeptide gene cluster contains three closely related sulfotransferases (Teg12, -13, and -14) that sulfate teicoplanin-like glycopeptides at three unique sites. Here we report a series of structures: an apo structure of Teg12, Teg12 bound to the desulfated cosubstrate 3{prime}-phosphoadenosine 5{prime}-phosphate, and Teg12 bound to the teicoplanin aglycone. Teg12 appears to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding, and catalysis. Loop regions that exhibit the most conformational flexibility show the least sequence conservation between TEG sulfotransferases. Site-directed mutagenesis guided by our structural studies confirmed the importance of key catalytic residues as well as the importance of residues found throughout the conformationally flexible loop regions.

  17. Human phenol sulfotransferases hP-PST and hM-PST activate propane 2-nitronate to a genotoxicant.

    Science.gov (United States)

    Kreis, P; Brandner, S; Coughtrie, M W; Pabel, U; Meinl, W; Glatt, H; Andrae, U

    2000-02-01

    The industrial solvent 2-nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound in rats has been attributed to sulfotransferase-mediated formation of DNA-reactive nitrenium ions from the anionic form of 2-NP, propane 2-nitronate (P2N). Whether human sulfotransferases are capable of activating P2N is unknown. In the present study we have addressed this question by investigating the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of human sulfotransferases, the phenol-sulfating and the monoamine-sulfating phenol sulfotransferases (hP-PST and hM-PST) and the human hydroxysteroid sulfotransferase (hHST). Genotoxicity was assessed by measuring the induction of DNA repair synthesis and by analyzing the formation of DNA modifications. P2N induced repair synthesis in V79-hP-PST and V79-hM-PST cells, whereas induction of repair synthesis in V79-hHST cells was negligible. P2N also resulted in the formation of 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine in V79-hP-PST cells, but not in the parental V79-MZ cells, which do not show any sulfotransferase activity. Acetone oxime, the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results show that the human phenol sulfotransferases P-PST and M-PST are capable of metabolically activating P2N (P-PST > M-PST) and that the underlying mechanism is apparently identical to that resulting in the activation of P2N in rat liver, where 2-NP causes carcinomas. These results support the notion that 2-NP should be regarded as a potential human carcinogen.

  18. Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3'-Phosphoadenosine 5'-Phosphosulfate and ATP.

    Science.gov (United States)

    Mi-ichi, Fumika; Nozawa, Akira; Yoshida, Hiroki; Tozawa, Yuzuru; Nozaki, Tomoyoshi

    2015-11-01

    Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3'-phosphoadenosine 5'-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids. Copyright © 2015 Mi-ichi et al.

  19. Cholesterol Sulfate and Cholesterol Sulfotransferase Inhibit Gluconeogenesis by Targeting Hepatocyte Nuclear Factor 4α

    Science.gov (United States)

    Shi, Xiongjie; Cheng, Qiuqiong; Xu, Leyuan; Yan, Jiong; Jiang, Mengxi; He, Jinhan; Xu, Meishu; Stefanovic-Racic, Maja; Sipula, Ian; O'Doherty, Robert Martin; Ren, Shunlin

    2014-01-01

    Sulfotransferase (SULT)-mediated sulfation represents a critical mechanism in regulating the chemical and functional homeostasis of endogenous and exogenous molecules. The cholesterol sulfotransferase SULT2B1b catalyzes the sulfoconjugation of cholesterol to synthesize cholesterol sulfate (CS). In this study, we showed that the expression of SULT2B1b in the liver was induced in obese mice and during the transition from the fasted to the fed state, suggesting that the regulation of SULT2B1b is physiologically relevant. CS and SULT2B1b inhibited gluconeogenesis by targeting the gluconeogenic factor hepatocyte nuclear factor 4α (HNF4α) in both cell cultures and transgenic mice. Treatment of mice with CS or transgenic overexpression of the CS-generating enzyme SULT2B1b in the liver inhibited hepatic gluconeogenesis and alleviated metabolic abnormalities both in mice with diet-induced obesity (DIO) and in leptin-deficient (ob/ob) mice. Mechanistically, CS and SULT2B1b inhibited gluconeogenesis by suppressing the expression of acetyl coenzyme A (acetyl-CoA) synthetase (Acss), leading to decreased acetylation and nuclear exclusion of HNF4α. Our results also suggested that leptin is a potential effector of SULT2B1b in improving metabolic function. We conclude that SULT2B1b and its enzymatic by-product CS are important metabolic regulators that control glucose metabolism, suggesting CS as a potential therapeutic agent and SULT2B1b as a potential therapeutic target for metabolic disorders. PMID:24277929

  20. Enzymatic sulfation of steroids. XI. The extensive purification and some properties of hepatic sulfotransferase III from female rats.

    Science.gov (United States)

    Singer, S S; Bruns, L

    1980-08-01

    Our earlier studies showed that livers from female rats contained three glucocorticoid sulfating enzymes we named sulfotransferases I, II, and III, (STI, STII, and STIII, respectively). In this report STIII from female Charles River CD rats was purified 1010- to 1300-fold compared with liver homogenates. The most highly purified STIII fraction electrophoresed as a single protein band. The molecular weight of STIII was 68 300 +/- 4900. Its pH optimum for cortisol sulfation was pH 6.0 +/- 0.1. However, it was routinely assayed at pH 6.8 for reasons enumerated in the text. Cortisol sulfation by STIII proceeded by either an ordered sequential mechanism or by an Iso Theorell-Chance mechanism at pH 6.8. The Km's for cortisol and the reaction coenzyme, 3'-phosphoadenosine-5'-phosphosulfate were 6.48 +/- 0.78 and 6.78 +/- 1.26 microM, respectively. Comparison of the ability of the enzyme to sulfate 40 microM cortisol, estradiol-17 beta, testosterone, deoxycorticosterone, and dehydroepiandrosterone, showed that the glucocorticoid was sulfated preferentially. Interestingly, its cortisol sulfotransferase activity was inhibited by a number of steroids. p-Hydroxymercuribenzoate inhibition studies indicated the presence of essential sulfhydryl groups in STIII. The enzyme was activated by divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts. It was inactivated by Zn2+ and Cd2+ salts. The text compares STIII from female rats with other steroid sulfotransferases including the major glucocorticoid sulfotransferase from male rats.

  1. Germline ablation of dermatan-4O-sulfotransferase1 reduces regeneration after mouse spinal cord injury.

    Science.gov (United States)

    Rost, S; Akyüz, N; Martinovic, T; Huckhagel, T; Jakovcevski, I; Schachner, M

    2016-01-15

    Chondroitin/dermatan sulfate proteoglycans (CSPGs/DSPGs) are major components of the extracellular matrix. Their expression is generally upregulated after injuries to the adult mammalian central nervous system, which is known for its low ability to restore function after injury. Several studies support the view that CSPGs inhibit regeneration after injury, whereas the functions of DSPGs in injury paradigms are less certain. To characterize the functions of DSPGs in the presence of CSPGs, we studied young adult dermatan-4O-sulfotransferase1-deficient (Chst14(-/-)) mice, which express chondroitin sulfates (CSs), but not dermatan sulfates (DSs), to characterize the functional outcome after severe compression injury of the spinal cord. In comparison to their wild-type (Chst14(+/+)) littermates, regeneration was reduced in Chst14(-/-) mice. No differences between genotypes were seen in the size of spinal cords, numbers of microglia and astrocytes neither in intact nor injured spinal cords after injury. Monoaminergic innervation and re-innervation of the spinal cord caudal to the lesion site as well as expression levels of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were similar in both genotypes, independent of whether they were injured and examined 6weeks after injury or not injured. These results suggest that, in contrast to CSPGs, DSPGs, being the products of Chst14 enzymatic activity, promote regeneration after injury of the adult mouse central nervous system. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. In vivo induction of tyrosylprotein sulfotransferase by ethanol: role of increased enzyme synthesis.

    Science.gov (United States)

    Ramaprasad, P; Kasinathan, C

    1998-08-01

    Tyrosine sulfation is a posttranslational modification involved in the synthesis, secretion, and biological activity of proteins and peptides. Our previous studies have demonstrated that the enzyme activity was induced by ethanol. In the present work, the induction was studied in detail. Initial experiments were conducted to examine the time course of tyrosylprotein sulfotransferase (TPST) induction in rats pair-fed liquid diets containing either ethanol or carbohydrate substitute (controls). Marked elevation of TPST activity (3-fold) was measured on day 10 in the liver and gastric mucosa of ethanol-fed rats. Ethanol-mediated enhancement was also noticed by Western-blot analysis with anti-TPST antibody in both the liver and gastric mucosa on days 5 and 10. We then determined the steady-state TPST protein turnover in ethanol-fed and control animals that were given 35S-methionine after 10 days of pair-feeding with liquid diet. The rates of TPST synthesis assessed by measuring initial rates of incorporation of 35S-methionine into TPST was increased in the liver and gastric mucosa of animals fed with ethanol. Monophasic exponential decay curves showed that TPST protein half-lives for liver (control: 34 hr, ethanol: 32 hr) and gastric mucosa (control: 52 hr, ethanol: 48 hr) did not differ between control and ethanol groups. Our overall results indicate that the in vivo induction of TPST by ethanol involves increased enzyme synthesis rather than decreased enzyme degradation.

  3. Sulfotransferase-independent genotoxicity of illudin S and its acylfulvene derivatives in bacterial and mammalian cells.

    Science.gov (United States)

    Glatt, Hansruedi; Pietsch, Kathryn E; Sturla, Shana J; Meinl, Walter

    2014-01-01

    Acylfulvenes are a class of antitumor agents derived from illudin S, a sesquiterpenoid toxin isolated from mushrooms of the genus Omphalotus. Although DNA appears to be their major target, no data concerning mutagenicity of acylfulvenes are available in the literature, and limited data have been published on illudin S. Enzyme-mediated biotransformations have been demonstrated to influence the cytotoxicity of acylfulvenes. Illudin S and some acylfulvenes [e.g., (-)-6-hydroxymethylacylfulvene (HMAF)] are allylic alcohols with potential for enhanced cytotoxicity and genotoxicity by means of metabolic sulfation. Therefore, we studied the influence of various heterologously expressed human sulfotransferases (SULTs) on biological activities of illudin S and HMAF in bacterial and mammalian cells. (-)-Acylfulvene (AF) was tested as a congener lacking an allylic hydroxyl group. We found: (1) all three compounds were mutagenic in standard Salmonella typhimurium strains TA98, TA100 and TA104; (2) they induced gene mutations (at the hypoxanthine phosphoribosyl transferase locus) and sister chromatid exchange (SCE) in Chinese hamster V79 cells; (3) these effects were practically unaffected when human SULTs were expressed in the target bacteria or mammalian cells (using SCE as the endpoint); (4) illudin S demonstrated 40-600 times higher genotoxic activities than the semisynthetic acylfulvenes studied; it was positive in the SCE test even at a concentration of 0.3 nM; (5) genotoxicity in mammalian cells was observed at substantially lower concentrations of the compounds than required for a positive result in the bacterial test (400 nM with illudin S). We conclude that illudin S, HMAF and AF are potent genotoxicants and human SULTs do not play a significant role in their bioactivation.

  4. Cytosolic delivery of materials with endosome-disrupting colloids

    Science.gov (United States)

    Helms, Brett A.; Bayles, Andrea R.

    2016-03-15

    A facile procedure to deliver nanocrystals to the cytosol of live cells that is both rapid and general. The technique employs a unique cationic core-shell polymer colloid that directs nanocrystals to the cytosol of living cells within a few hours of incubation. The present methods and compositions enable a host of advanced applications arising from efficient cytosolic delivery of nanocrystal imaging probes: from single particle tracking experiments to monitoring protein-protein interactions in live cells for extended periods.

  5. Sulfotransferase activity in plucked hair follicles predicts response to topical minoxidil in the treatment of female androgenetic alopecia.

    Science.gov (United States)

    Roberts, Janet; Desai, Nisha; McCoy, John; Goren, Andy

    2014-01-01

    Two percent topical minoxidil is the only US Food and Drug Administration-approved drug for the treatment of female androgenetic alopecia (AGA). Its success has been limited by the low percentage of responders. Meta-analysis of several studies reporting the number of responders to 2% minoxidil monotherapy indicates moderate hair regrowth in only 13-20% of female patients. Five percent minoxidil solution, when used off-label, may increase the percentage of responders to as much as 40%. As such, a biomarker for predicting treatment response would have significant clinical utility. In a previous study, Goren et al. reported an association between sulfotransferase activity in plucked hair follicles and minoxidil response in a mixed cohort of male and female patients. The aim of this study was to replicate these findings in a well-defined cohort of female patients with AGA treated with 5% minoxidil daily for a period of 6 months. Consistent with the prior study, we found that sulfotransferase activity in plucked hair follicles predicts treatment response with 93% sensitivity and 83% specificity. Our study further supports the importance of minoxidil sulfation in eliciting a therapeutic response and provides further insight into novel targets for increasing minoxidil efficacy. © 2014 Wiley Periodicals, Inc.

  6. Congenital joint dislocations caused by carbohydrate sulfotransferase 3 deficiency in recessive Larsen syndrome and humero-spinal dysostosis.

    Science.gov (United States)

    Hermanns, Pia; Unger, Sheila; Rossi, Antonio; Perez-Aytes, Antonio; Cortina, Hector; Bonafé, Luisa; Boccone, Loredana; Setzu, Valeria; Dutoit, Michel; Sangiorgi, Luca; Pecora, Fabio; Reicherter, Kerstin; Nishimura, Gen; Spranger, Jürgen; Zabel, Bernhard; Superti-Furga, Andrea

    2008-06-01

    Deficiency of carbohydrate sulfotransferase 3 (CHST3; also known as chondroitin-6-sulfotransferase) has been reported in a single kindred so far and in association with a phenotype of severe chondrodysplasia with progressive spinal involvement. We report eight CHST3 mutations in six unrelated individuals who presented at birth with congenital joint dislocations. These patients had been given a diagnosis of either Larsen syndrome (three individuals) or humero-spinal dysostosis (three individuals), and their clinical features included congenital dislocation of the knees, elbow joint dysplasia with subluxation and limited extension, hip dysplasia or dislocation, clubfoot, short stature, and kyphoscoliosis developing in late childhood. Analysis of chondroitin sulfate proteoglycans in dermal fibroblasts showed markedly decreased 6-O-sulfation but enhanced 4-O-sulfation, confirming functional impairment of CHST3 and distinguishing them from diastrophic dysplasia sulphate transporter (DTDST)-deficient cells. These observations provide a molecular basis for recessive Larsen syndrome and indicate that recessive Larsen syndrome, humero-spinal dysostosis, and spondyloepiphyseal dysplasia Omani type form a phenotypic spectrum.

  7. Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

    Science.gov (United States)

    Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

    2017-08-04

    Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Cloning and characterization of human liver cytosolic beta-glycosidase

    NARCIS (Netherlands)

    De Graaf, M; Van Veen, IC; Van Der Meulen-Muileman, IH; Gerritsen, WR; Pinedo, HM; Haisma, HJ

    2001-01-01

    Cytosolic beta -glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of beta -D-glycosides. including beta -D-glucoside acid beta -D-galactoside. We therefore refer to this enzyme as cytosolic beta

  9. Quantitative assessment of cytosolic Salmonella in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Leigh A Knodler

    Full Text Available Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV. We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1, but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.

  10. The Arabidopsis cytosolic proteome

    DEFF Research Database (Denmark)

    Ito, Jun; Parsons, Harriet Tempé; Heazlewood, Joshua L.

    2014-01-01

    of secondary metabolites, and accumulation of enzymes for defense and detoxification. This central role is highlighted by estimates indicating that the majority of eukaryotic proteins are cytosolic. Arabidopsis thaliana has been the subject of numerous proteomic studies on its different subcellular...... compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted...

  11. Induction of PNAd and N-acetylglucosamine 6-O-sulfotransferases 1 and 2 in mouse collagen-induced arthritis

    Directory of Open Access Journals (Sweden)

    Rosen Steven D

    2006-06-01

    Full Text Available Abstract Background Leukocyte recruitment across blood vessels is fundamental to immune surveillance and inflammation. Lymphocyte homing to peripheral lymph nodes is mediated by the adhesion molecule, L-selectin, which binds to sulfated carbohydrate ligands on high endothelial venules (HEV. These glycoprotein ligands are collectively known as peripheral node addressin (PNAd, as defined by the function-blocking monoclonal antibody known as MECA-79. The sulfation of these ligands depends on the action of two HEV-expressed N-acetylglucosamine 6-O-sulfotransferases: GlcNAc6ST-2 and to a lesser degree GlcNAc6ST-1. Induction of PNAd has also been shown to occur in a number of human inflammatory diseases including rheumatoid arthritis (RA. Results In order to identify an animal model suitable for investigating the role of PNAd in chronic inflammation, we examined the expression of PNAd as well as GlcNAc6ST-1 and -2 in collagen-induced arthritis in mice. Here we show that PNAd is expressed in the vasculature of arthritic synovium in mice immunized with collagen but not in the normal synovium of control animals. This de novo expression of PNAd correlates strongly with induction of transcripts for both GlcNAc6ST-1 and GlcNAc6ST-2, as well as the expression of GlcNAc6ST-2 protein. Conclusion Our results demonstrate that PNAd and the sulfotransferases GlcNAc6ST-1 and 2 are induced in mouse collagen-induced arthritis and suggest that PNAd antagonists or inhibitors of the enzymes may have therapeutic benefit in this widely-used mouse model of RA.

  12. SwissProt search result: AK071840 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071840 J023121F12 (O35400) Sulfotransferase family cytosolic 2B member 1 (EC 2.8.2.2) (Sulfotransfer...ase 2B1) (Sulfotransferase 2B) (Alcohol sulfotransferase) (Hydroxysteroid sulfotransferase 2) ST2B1_MOUSE 2e-18 ...

  13. SwissProt search result: AK105239 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105239 001-113-B04 (O35400) Sulfotransferase family cytosolic 2B member 1 (EC 2.8.2.2) (Sulfotransfer...ase 2B1) (Sulfotransferase 2B) (Alcohol sulfotransferase) (Hydroxysteroid sulfotransferase 2) ST2B1_MOUSE 4e-20 ...

  14. SwissProt search result: AK241143 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241143 J065111M09 (O35400) Sulfotransferase family cytosolic 2B member 1 (EC 2.8.2.2) (Sulfotransfer...ase 2B1) (Sulfotransferase 2B) (Alcohol sulfotransferase) (Hydroxysteroid sulfotransferase 2) ST2B1_MOUSE 2e-19 ...

  15. SwissProt search result: AK105552 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105552 001-128-A12 (O35400) Sulfotransferase family cytosolic 2B member 1 (EC 2.8.2.2) (Sulfotransfer...ase 2B1) (Sulfotransferase 2B) (Alcohol sulfotransferase) (Hydroxysteroid sulfotransferase 2) ST2B1_MOUSE 6e-22 ...

  16. SwissProt search result: AK061204 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061204 006-210-B05 (O35400) Sulfotransferase family cytosolic 2B member 1 (EC 2.8.2.2) (Sulfotransfer...ase 2B1) (Sulfotransferase 2B) (Alcohol sulfotransferase) (Hydroxysteroid sulfotransferase 2) ST2B1_MOUSE 6e-20 ...

  17. SwissProt search result: AK243653 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243653 J100088J11 (O35400) Sulfotransferase family cytosolic 2B member 1 (EC 2.8.2.2) (Sulfotransfer...ase 2B1) (Sulfotransferase 2B) (Alcohol sulfotransferase) (Hydroxysteroid sulfotransferase 2) ST2B1_MOUSE 5e-25 ...

  18. SwissProt search result: AK058698 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058698 001-019-C01 (O35400) Sulfotransferase family cytosolic 2B member 1 (EC 2.8.2.2) (Sulfotransfer...ase 2B1) (Sulfotransferase 2B) (Alcohol sulfotransferase) (Hydroxysteroid sulfotransferase 2) ST2B1_MOUSE 2e-21 ...

  19. SwissProt search result: AK066828 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066828 J013075L15 (O35400) Sulfotransferase family cytosolic 2B member 1 (EC 2.8.2.2) (Sulfotransfer...ase 2B1) (Sulfotransferase 2B) (Alcohol sulfotransferase) (Hydroxysteroid sulfotransferase 2) ST2B1_MOUSE 6e-20 ...

  20. SwissProt search result: AK059900 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059900 006-208-G02 (O35400) Sulfotransferase family cytosolic 2B member 1 (EC 2.8.2.2) (Sulfotransfer...ase 2B1) (Sulfotransferase 2B) (Alcohol sulfotransferase) (Hydroxysteroid sulfotransferase 2) ST2B1_MOUSE 1e-17 ...

  1. The Arabidopsis cytosolic proteome: the metabolic heart of the cell

    Directory of Open Access Journals (Sweden)

    Jun eIto

    2014-02-01

    Full Text Available The plant cytosol is the major intracellular fluid that acts as the medium for inter-organellar crosstalk and where a plethora of important biological reactions take place. These include its involvement in protein synthesis and degradation, stress response signaling, carbon metabolism, biosynthesis of secondary metabolites and accumulation of enzymes for defense and detoxification. This central role is highlighted by estimates indicating that the majority of eukaryotic proteins are cytosolic. Arabidopsis thaliana has been the subject of numerous proteomic studies on its different subcellular compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted proteomic characterizations of complexes is included. Despite this, few groups are currently applying advanced proteomic approaches to this important metabolic space. This review will highlight the current state of the Arabidopsis cytosolic proteome since its initial characterization a few years ago.

  2. Cytosolic proteostasis through importing of misfolded proteins into mitochondria.

    Science.gov (United States)

    Ruan, Linhao; Zhou, Chuankai; Jin, Erli; Kucharavy, Andrei; Zhang, Ying; Wen, Zhihui; Florens, Laurence; Li, Rong

    2017-03-16

    Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.

  3. PPARγ regulates expression of carbohydrate sulfotransferase 11 (CHST11/C4ST1, a regulator of LPL cell surface binding.

    Directory of Open Access Journals (Sweden)

    Ismayil Tasdelen

    Full Text Available The transcription factor PPARγ is the key regulator of adipocyte differentiation, function and maintenance, and the cellular target of the insulin-sensitizing thiazolidinediones. Identification and functional characterization of genes regulated by PPARγ will therefore lead to a better understanding of adipocyte biology and may also contribute to the development of new anti-diabetic drugs. Here, we report carbohydrate sulfotransferase 11 (Chst11/C4st1 as a novel PPARγ target gene. Chst11 can sulphate chondroitin, a major glycosaminoglycan involved in development and disease. The Chst11 gene contains two functional intronic PPARγ binding sites, and is up-regulated at the mRNA and protein level during 3T3-L1 adipogenesis. Chst11 knockdown reduced intracellular lipid accumulation in mature adipocytes, which is due to a lowered activity of lipoprotein lipase, which may associate with the adipocyte cell surface through Chst11-mediated sulfation of chondroitin, rather than impaired adipogenesis. Besides directly inducing Lpl expression, PPARγ may therefore control lipid accumulation by elevating the levels of Chst11-mediated proteoglycan sulfation and thereby increasing the binding capacity for Lpl on the adipocyte cell surface.

  4. A variation in the cerebroside sulfotransferase gene is linked to exercise-modified insulin resistance and to type 2 diabetes

    DEFF Research Database (Denmark)

    Roeske-Nielsen, A.; Buschard, K.; Manson, J.E.

    2009-01-01

    .11). The aim of this study was to investigate whether two single nucleotide polymorphisms (SNP), rs2267161 located in an exon or rs42929 located in an intron, in the gene encoding CST are linked to type 2 diabetes (T2D). METHODS: As a population survey, 265 male and female patients suffering from T2D and 291......, the homozygous CC individuals displayed lower insulin resistance measured by HOMA-IR (P = .05) than the C/T or TT persons; this was particularly prevalent in individuals who exercise (P = .03). CONCLUSION: Heterozygosity at SNP rs2267161 in the gene encoding the CST enzyme confers increased risk of T2D. Females......AIMS: The glycosphingolipid beta-galactosylceramide-3-O-sulfate (sulfatide) is present in the secretory granules of the insulin producing beta-cells and may act as a molecular chaperone of insulin. The final step in sulfatide synthesis is performed by cerebroside sulfotransferase (CST) (EC 2.8.2...

  5. Targeted liposomes for cytosolic drug delivery to tumor cells

    NARCIS (Netherlands)

    Mastrobattista, E.

    2001-01-01

    In this thesis, a Trojan horse strategy with antibody-targeted liposomes has been followed to obtain cytosolic delivery of biotherapeutics to tumor cells in vitro. This strategy involves targeting of immunoliposomes to specific receptors on tumor cells that result in receptor-mediated uptake of the

  6. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

    Science.gov (United States)

    Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

    2017-08-01

    One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

  7. Calcium-independent phospholipase A2 in rat tissue cytosols

    NARCIS (Netherlands)

    Pierik, A.J.; Nijssen, J.G.; Aarsman, A.J.; Bosch, H. van den

    1988-01-01

    Cytosols (105000 X g supernatant) from seven rat tissues were assayed for Ca²⁺-independent phospholipase A₂ activity with either 1-acyl-2-[1-¹⁴C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[l-¹⁴C]linoleoyl-snglycero- 3-phosphoethanohunine or

  8. Monitoring disulfide bond formation in the eukaryotic cytosol

    DEFF Research Database (Denmark)

    Østergaard, Henrik; Tachibana, Christine; Winther, Jakob R.

    2004-01-01

    Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green flu...

  9. Genomic organization of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene: Exclusion from a causative role in the pathogenesis of Treacher Collins syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Gladwin, A.J.; Dixon, J.; Loftus, S.K.; Wasmuth, J.J.; Dixon, M.J. [Univ. of Manchester (United Kingdom)]|[Univ. of California, Irvine, CA (United States)

    1996-03-05

    Heparan sulfate-N-deacetylase/N-sulfotransferase (HSST) catalyzes both the N-deacetylation and the N-sulfation of heparan sulfate. Previous studies have resulted in the isolation of the human HSST gene from within the Treacher Collins syndrome locus (TCOF1) critical region on 5q. In the present study, the genomic organization of the HSST gene has been elucidated, and the 14 exons identified have been tested for TCOF1-specific mutations. As a result of these studies, mutations within the coding sequence and adjacent splice junctions of HSST can be excluded from a causative role in the pathogenesis of Treacher Collins syndrome. 13 refs., 1 fig., 2 tabs.

  10. Hydroxysteroid sulfotransferase SULT2B1b promotes hepatocellular carcinoma cells proliferation in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Xiaoming Yang

    Full Text Available Hydroxysteroid sulfotransferase 2B1b (SULT2B1b is highly selective for the addition of sulfate groups to 3β-hydroxysteroids. Although previous reports have suggested that SULT2B1b is correlated with cell proliferation of hepatocytes, the relationship between SULT2B1b and the malignant phenotype of hepatocarcinoma cells was not clear. In the present study, we found that SULT2B1 was comparatively higher in the human hepatocarcinoma tumorous tissues than their adjacent tissues. Besides, SULT2B1b overexpression promoted the growth of the mouse hepatocarcinoma cell line Hepa1-6, while Lentivirus-mediated SULT2B1b interference inhibited growth as assessed by the CCK-8 assay. Likewise, inhibition of SULT2B1b expression induced cell-cycle arrest and apoptosis in Hepa1-6 cells by upregulating the expression of FAS, downregulating the expression of cyclinB1, BCL2 and MYC in vitro and in vivo at both the transcript and protein levels. Knock-down of SULT2B1b expression significantly suppressed tumor growth in nude mouse xenografts. Moreover, proliferation rates and SULT2B1b expression were highly correlated in the human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721 and BEL-7402 cells. Knock-down of SULT2B1b inhibited cell growth and cyclinB1 levels in human hepatocarcinoma cells and suppressed xenograft growth in vivo. In conclusion, SULT2B1b expression promotes proliferation of hepatocellular carcinoma cells in vitro and in vivo, which may contribute to the progression of HCC.

  11. Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein

    DEFF Research Database (Denmark)

    Liu, Xiangdong; Martens, Helle; Schulz, Alexander

    Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein.......Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein....

  12. Amination of tyrosine in liver cytosol protein of male F344 rats treated with 2-nitropropane, 2-nitrobutane, 3-nitropentane, or acetoxime.

    Science.gov (United States)

    Sodum, R S; Fiala, E S

    1997-12-01

    Previously, the secondary nitroalkane 2-nitropropane, a strong hepatocarcinogen in rats, had been shown to induce the formation of 8-aminoguanine in both DNA and RNA of rat liver through a sulfotransferase-mediated pathway. This pathway was postulated to convert the carcinogen into an aminating species [Sodum, R. S., et al. (1994) Chem. Res. Toxicol. 7, 344-351]. To submit this postulate to further test, we examined liver proteins of rats treated with 2-nitropropane, other carcinogenic secondary nitroalkanes, or the related rat liver tumorigen acetoxime for the presence of 3-aminotyrosine, the expected product of tyrosine amination. Using ion-pair and/or cation-exchange high-performance liquid chromatography with electrochemical detection, we found that the liver cytosolic proteins of these animals contained 0.1-1.5 mol of 3-aminotyrosine/10(3) mol of tyrosine. Treatment with the noncarcinogenic primary nitroalkane 1-nitropropane or with other primary nitroalkanes did not produce an analogous increase in the aminated amino acid (level of detection estimated at approximately 0.01 mol/10(3) mol of tyrosine). To our knowledge, this is the first report of the modification of protein tyrosine in vivo by a carcinogen. In vitro studies with acetoxime-O-sulfonate and hydroxylamine-O-sulfonate showed that these proposed intermediates in the activation pathway of 2-nitropropane react with guanosine to give 8-aminoguanosine, N1-aminoguanosine, and 8-oxoguanosine and also react with tyrosine to give 3-aminotyrosine and 3-hydroxytyrosine. The in vitro amination and oxidation of guanosine at C8 were also produced by acetophenoxime-O-sulfonate and 2-heptanoxime-O-sulfonate. These results provide additional evidence for the production of a reactive species capable of aminating nucleic acids and proteins from 2-nitropropane and other carcinogenic secondary nitroalkanes by a pathway involving oxime- and hydroxylamine-O-sulfonates as intermediates.

  13. Artificial Loading of ASC Specks with Cytosolic Antigens.

    Directory of Open Access Journals (Sweden)

    Ali Can Sahillioğlu

    Full Text Available Inflammasome complexes form upon interaction of Nod Like Receptor (NLR proteins with pathogen associated molecular patterns (PAPMS inside the cytosol. Stimulation of a subset of inflammasome receptors including NLRP3, NLRC4 and AIM2 triggers formation of the micrometer-sized spherical supramolecular complex called the ASC speck. The ASC speck is thought to be the platform of inflammasome activity, but the reason why a supramolecular complex is preferred against oligomeric platforms remains elusive. We observed that a set of cytosolic proteins, including the model antigen ovalbumin, tend to co-aggregate on the ASC speck. We suggest that co-aggregation of antigenic proteins on the ASC speck during intracellular infection might be instrumental in antigen presentation.

  14. DMPD: Cytosolic DNA recognition for triggering innate immune responses. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18280611 Cytosolic DNA recognition for triggering innate immune responses. Takaoka ...A, Taniguchi T. Adv Drug Deliv Rev. 2008 Apr 29;60(7):847-57. Epub 2007 Dec 31. (.png) (.svg) (.html) (.csml) Show Cytosolic DNA reco...gnition for triggering innate immune responses. PubmedID 18280611 Title Cytosolic DNA reco

  15. A new view of the bacterial cytosol environment.

    Directory of Open Access Journals (Sweden)

    Benjamin P Cossins

    2011-06-01

    Full Text Available The cytosol is the major environment in all bacterial cells. The true physical and dynamical nature of the cytosol solution is not fully understood and here a modeling approach is applied. Using recent and detailed data on metabolite concentrations, we have created a molecular mechanical model of the prokaryotic cytosol environment of Escherichia coli, containing proteins, metabolites and monatomic ions. We use 200 ns molecular dynamics simulations to compute diffusion rates, the extent of contact between molecules and dielectric constants. Large metabolites spend ∼80% of their time in contact with other molecules while small metabolites vary with some only spending 20% of time in contact. Large non-covalently interacting metabolite structures mediated by hydrogen-bonds, ionic and π stacking interactions are common and often associate with proteins. Mg(2+ ions were prominent in NIMS and almost absent free in solution. Κ(+ is generally not involved in NIMSs and populates the solvent fairly uniformly, hence its important role as an osmolyte. In simulations containing ubiquitin, to represent a protein component, metabolite diffusion was reduced owing to long lasting protein-metabolite interactions. Hence, it is likely that with larger proteins metabolites would diffuse even more slowly. The dielectric constant of these simulations was found to differ from that of pure water only through a large contribution from ubiquitin as metabolite and monatomic ion effects cancel. These findings suggest regions of influence specific to particular proteins affecting metabolite diffusion and electrostatics. Also some proteins may have a higher propensity for associations with metabolites owing to their larger electrostatic fields. We hope that future studies may be able to accurately predict how binding interactions differ in the cytosol relative to dilute aqueous solution.

  16. Yeast and Mammals Utilize Similar Cytosolic Components to Drive Protein Transport through the Golgi Complex

    Science.gov (United States)

    Dunphy, William G.; Pfeffer, Suzanne R.; Clary, Douglas O.; Wattenberg, Binks W.; Glick, Benjamin S.; Rothman, James E.

    1986-03-01

    Vesicular transport between successive compartments of the mammalian Golgi apparatus has recently been reconstituted in a cell-free system. In addition to ATP, transport requires both membrane-bound and cytosolic proteins. Here we report that the cytosol fraction from yeast will efficiently substitute for mammalian cytosol. Mammalian cytosol contains several distinct transport factors, which we have distinguished on the basis of gel filtration and ion-exchange chromatography. Yeast cytosol appears to contain the same collection of transport factors. Resolved cytosol factors from yeast and mammals complement each other in a synergistic manner. These findings suggest that the molecular mechanisms of intracellular protein transport have been conserved throughout evolution. Moreover, this hybrid cell-free system will enable the application of yeast genetics to the identification and isolation of cytosolic proteins that sustain intracellular protein transport.

  17. Spectral components of cytosolic [Ca2+] spiking in neurons

    DEFF Research Database (Denmark)

    Kardos, J; Szilágyi, N; Juhász, G

    1998-01-01

    into evolutionary spectra of a characteristic set of frequencies. Non-delayed small spikes on top of sustained [Ca2+]c were synthesized by a main component frequency, 0.132+/-0.012 Hz, showing its maximal amplitude in phase with the start of depolarization (25 mM KCI) combined with caffeine (10 mM) application......We show here, by means of evolutionary spectral analysis and synthesis of cytosolic Ca2+ ([Ca2+]c) spiking observed at the single cell level using digital imaging fluorescence microscopy of fura-2-loaded mouse cerebellar granule cells in culture, that [Ca2+]c spiking can be resolved...

  18. Metabolism of antitumor acylfulvene by rat liver cytosol.

    Science.gov (United States)

    McMorris, T C; Elayadi, A N; Yu, J; Kelner, M J

    1999-01-01

    Illudins are novel compounds from which a potent class of antitumor agents, called acylfulvenes, have been synthesized. The model illudin, illudin S, has marked in vitro and in vivo toxicity but displays a poor therapeutic index. The toxicity of illudin S is believed to involve a combination of enzymatic reduction and chemical reaction. Enzymatic reduction by a cytosolic NADPH-dependent enzyme produces an aromatic metabolite, as does reaction with thiols. Acylfulvene is formed from illudin S by reverse Prins reaction. Acylfulvene is 100-fold less toxic in vitro and in vivo than illudin S but possesses marked antitumor efficacy in vivo, thus displaying opposite properties from illudin S. For this reason we investigated the in vitro metabolism of acylfulvene. Incubation of acylfulvene with NADPH and rat liver cytosol yielded two metabolites. One metabolite, the aromatic product, is similar to that obtained with illudin S in this in vitro system and was anticipated. The other metabolite, the hydroxylated product, was not expected and no corresponding metabolite for illudin S could be detected. The production of this hydroxylated metabolite from acylfulvene may explain, in part, the increased antitumor activity of novel acylfulvenes as compared with the illudins.

  19. Cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene from the Treacher Collins syndrome candidate region at 5q32-q33.1

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, J.; Loftus, S.K.; Gladwin, A.J. [Univ. of Manchester (United Kingdom)] [and others

    1995-03-20

    Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. Previous studies have shown that the Treacher Collins syndrome locus is flanked by D5S519 and SPARC, and a yeast artificial chromosome contig encompassing this {open_quotes}critical region{close_quotes} has been completed. In the current investigation a cosmid containing D5S519 has been used to screen a human placental cDNA library. This has resulted in the cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene. Two different mRNA species that have identical protein coding sequences but that differ in the size and sequence of the 3{prime} untranslated regions (3{prime}UTR) have been identified. The smaller species has a 3{prime}UTR of 1035 bp, whereas that of the larger is 4878 bp. 24 refs., 3 figs.

  20. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  1. Effect of sodium molybdate on the thyroxine-binding affinity of transport cytosol proteins.

    Science.gov (United States)

    Svobodová, V; Kraml, J; Schreiber, V

    1985-01-01

    The presence of sodium molybdate during tissue homogenization is known to increase the number of cytosol binding sites for glucocorticoids, progesterone, androgens and oestrogens. We wondered whether a phenomenon similar to this stabilization of steroid receptors would also occur in thyroxine-binding cytosol protein. We found that the presence of sodium molybdate (10 mmol/l) in rat adenohypophyseal cytosol increased its thyroxine-binding capacity by up to 96%. In the case of binding protein cytosol minus molybdate, Ka = 5.5 X 10(9) l.mol-1, whereas for cytosol plus molybdate Ka(1) = 6.0 X 10(9) l.mol-1 and Ka(2) = 3.0 X 10(10) l.mol-1. Cytosol prepared without molybdate did not contain a binding protein class with a higher Ka. The effect is stereo-specific and the LT4 bond is not displaced by DT4.

  2. Structural characterization of coatomer in its cytosolic state

    Directory of Open Access Journals (Sweden)

    Shengliu Wang

    2016-07-01

    Full Text Available Abstract Studies on coat protein I (COPI have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.

  3. Metabolism of antitumor hydroxymethylacylfulvene by rat liver cytosol.

    Science.gov (United States)

    McMorris, T C; Elayadi, A N; Yu, J; Hu, Y; Kelner, M J

    1999-09-01

    Acylfulvenes are a potent class of antitumor agents derived from illudin S, a fungal sesquiterpene. Illudin S possesses antitumor activity but has a poor therapeutic index. Acylfulvene is 100-fold less toxic against human lung adenocarcinoma cells than illudin S, but inhibits tumor growth in human xenografts, opposite to illudin S. An analog of acylfulvene, MGI 114 (hydroxymethylacylfulvene), shows much greater efficacy, producing complete tumor regression in xenograft models. MGI 114 is currently in phase II clinical trials. Cytotoxicity of MGI 114, like that of illudin S, is believed to involve both chemical reaction and enzymatic reduction. Enzymatic reduction by a cytosolic NADPH-dependent enzyme (from rat liver) produced an aromatic metabolite similar to that formed from illudin S. However, the reaction occurred more slowly. In addition, four new metabolites were isolated, two hydroxylated derivatives and two in which the primary allylic hydroxyl was replaced by hydride. All retained the reactive centers of the parent MGI 114.

  4. Nod-like receptors: cytosolic watchdogs for immunity against pathogens.

    Directory of Open Access Journals (Sweden)

    Jean-Claude Sirard

    2007-12-01

    Full Text Available In mammals, tissue-specific sets of pattern-recognition molecules, including Nod-like receptors (NLR, enable concomitant and sequential detection of microbial-associated molecular patterns from both the extracellular and intracellular microenvironment. Repressing and de-repressing the cytosolic surveillance machinery contributes to vital immune homeostasis and protective responses within specific tissues. Conversely, defective biology of NLR drives the development of recurrent infectious, autoimmune and/or inflammatory diseases by failing to mount barrier functions against pathogens, to tolerate commensals, and/or to instruct the adaptive immune response against microbes. Better decoding microbial strategies that are evolved to circumvent NLR sensing will provide clues for the development of rational therapies aimed at curing and/or preventing common and emerging immunopathologies.

  5. Cytosolic nucleic acid sensors and innate immune regulation.

    Science.gov (United States)

    Ori, Daisuke; Murase, Motoya; Kawai, Taro

    2017-03-04

    During viral and bacterial infections, pathogen-derived cytosolic nucleic acids are recognized by the intracellular RNA sensors retinoic acid-inducible gene I and melanoma-differentiated gene 5 and intracellular DNA sensors, including cyclic-di-GMP-AMP synthase, absent in melanoma 2, interferon (IFN)-gamma inducible protein 16, polymerase III, and so on. Binding of intracellular nucleic acids to these sensors activates downstream signaling cascades, resulting in the production of type I IFNs and pro-inflammatory cytokines to induce appropriate systematic immune responses. While these sensors also recognize endogenous nucleic acids and activate immune responses, they can discriminate between self- and non-self-nucleic acids. However, dysfunction of these sensors or failure of regulatory mechanisms causes aberrant activation of immune response and autoimmune disorders. In this review, we focus on how intracellular immune sensors recognize exogenous nucleic acids and activate the innate immune system, and furthermore, how autoimmune diseases result from dysfunction of these sensors.

  6. The Medicago truncatula DMI1 protein modulates cytosolic calcium signaling

    DEFF Research Database (Denmark)

    Peiter, Edgar; Sun, Jongho; Heckmann, Anne Birgitte Lau

    2007-01-01

    nodulation have been cloned in model legumes. Among them, Medicago truncatula DMI1 (DOESN'T MAKE INFECTIONS1) is required for the generation of nucleus-associated calcium spikes in response to the rhizobial signaling molecule Nod factor. DMI1 encodes a membrane protein with striking similarities...... to the Methanobacterium thermoautotrophicum potassium channel (MthK). The cytosolic C terminus of DMI1 contains a RCK (regulator of the conductance of K+) domain that in MthK acts as a calcium-regulated gating ring controlling the activity of the channel. Here we show that a dmi1 mutant lacking the entire C terminus acts...... as a dominant-negative allele interfering with the formation of nitrogen-fixing nodules and abolishing the induction of calcium spikes by the G-protein agonist Mastoparan. Using both the full-length DMI1 and this dominant-negative mutant protein we show that DMI1 increases the sensitivity of a sodium...

  7. Inhibiting cytosolic translation and autophagy improves health in mitochondrial disease.

    Science.gov (United States)

    Peng, Min; Ostrovsky, Julian; Kwon, Young Joon; Polyak, Erzsebet; Licata, Joseph; Tsukikawa, Mai; Marty, Eric; Thomas, Jeffrey; Felix, Carolyn A; Xiao, Rui; Zhang, Zhe; Gasser, David L; Argon, Yair; Falk, Marni J

    2015-09-01

    Mitochondrial respiratory chain (RC) disease therapies directed at intra-mitochondrial pathology are largely ineffective. Recognizing that RC dysfunction invokes pronounced extra-mitochondrial transcriptional adaptations, particularly involving dysregulated translation, we hypothesized that translational dysregulation is itself contributing to the pathophysiology of RC disease. Here, we investigated the activities, and effects from direct inhibition, of a central translational regulator (mTORC1) and its downstream biological processes in diverse genetic and pharmacological models of RC disease. Our data identify novel mechanisms underlying the cellular pathogenesis of RC dysfunction, including the combined induction of proteotoxic stress, the ER stress response and autophagy. mTORC1 inhibition with rapamycin partially ameliorated renal disease in B6.Pdss2(kd/kd) mice with complexes I-III/II-III deficiencies, improved viability and mitochondrial physiology in gas-1(fc21) nematodes with complex I deficiency, and rescued viability across a variety of RC-inhibited human cells. Even more effective was probucol, a PPAR-activating anti-lipid drug that we show also inhibits mTORC1. However, directly inhibiting mTORC1-regulated downstream activities yielded the most pronounced and sustained benefit. Partial inhibition of translation by cycloheximide, or of autophagy by lithium chloride, rescued viability, preserved cellular respiratory capacity and induced mitochondrial translation and biogenesis. Cycloheximide also ameliorated proteotoxic stress via a uniquely selective reduction of cytosolic protein translation. RNAseq-based transcriptome profiling of treatment effects in gas-1(fc21) mutants provide further evidence that these therapies effectively restored altered translation and autophagy pathways toward that of wild-type animals. Overall, partially inhibiting cytosolic translation and autophagy offer novel treatment strategies to improve health across the diverse array

  8. Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle

    DEFF Research Database (Denmark)

    Satrústegui, Jorgina; Bak, Lasse K

    2015-01-01

    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...

  9. Improving scFv antibody expression levels in the plant cytosol.

    NARCIS (Netherlands)

    Schouten, A.; Roosien, J.; Boer, de J.M.; Wilmink, A.; rosso, M.N.; Bosch, D.; Stiekema, W.J.; gommers, F.J.; Bakker, J.; Schots, A.

    1997-01-01

    Expression of single-chain antibody fragments (scFvs) in the plant cytosol is often cumbersome. It was unexpectedly shown that addition at the C-terminus of the ER retention signal KDEL resulted in significantly improved expression levels. In this report the cytosolic location of the scFv-CK was

  10. A quantitative comparison of cytosolic delivery via different protein uptake systems

    NARCIS (Netherlands)

    Verdurmen, W.P.R.; Mazlami, M.; Pluckthun, A.

    2017-01-01

    Over many years, a variety of delivery systems have been investigated that have the capacity to shuttle macromolecular cargoes, especially proteins, into the cytosol. Due to the lack of an objective way to quantify cytosolic delivery, relative delivery efficiencies of the various transport systems

  11. Cytosolic Ca2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary.

    Science.gov (United States)

    Thomas, P; Surprenant, A; Almers, W

    1990-11-01

    We have monitored cytosolic [Ca2+] with fura-2 and exocytosis by measuring the membrane capacitance, and we have studied the influence of cytosolic [Ca2+] on secretion in single endocrine cells. As in neurons, cytosolic Ca2+ is sufficient to trigger exocytosis. The rate of secretion grows with the fourth or fifth power of cytosolic [Ca2+], and paired stimuli reveal facilitation. Ca2+ influx through voltage-sensitive Ca2+ channels can stimulate secretion 1000-fold over the basal levels measured biochemically. Unlike neurons, however, melanotrophs continue to secrete for seconds afer a depolarizing pulse, while they extrude or sequester the Ca2+ that has entered through Ca2+ channels. Following episodes of secretion, pituitary cells can retrieve membrane with half-times around 30 s at 32 degrees C, even in the absence of cytosolic K+.

  12. Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

    Directory of Open Access Journals (Sweden)

    William A. McEwan

    2016-11-01

    Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

  13. Target proteins of the cytosolic thioredoxin in Plasmodium falciparum.

    Science.gov (United States)

    Kawazu, Shin-ichiro; Takemae, Hitoshi; Komaki-Yasuda, Kanako; Kano, Shigeyuki

    2010-06-01

    The target proteins of a cytosolic Trx (PfTrx-1) in Plasmodium falciparum with Trx-affinity chromatography were examined. Based on the Trx protein reduction pathway, we generated a cysteine mutant of PfTrx-1, which captures the target protein as a mixed disulfide intermediate. A number of proteins were captured with PfTrx-1(C33S) immobilized on resin and were eluted by DTT treatment. The PfTrx-1(C33S) immobilized resin-captured proteins were trypsin-digested and analyzed on a liquid chromatography-mass spectrometry system. Analysis of the sequence data against databases assigned 20 proteins, four of which had been found previously in P. falciparum, with the remaining 16 being new targets. The potential Trx-target proteins included those in pathways such as the redox cycle, protein biosynthesis, energy metabolism and signal transduction. We captured 4 enzymes in the glycolysis pathway (hexokinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase and L-lactate dehydrogenase (LDH)) as Trx-targets, and we found that PfTrx-1 enhanced the activity of PfGAPDH and PfLDH.

  14. Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

    Science.gov (United States)

    Mellouk, Nora; Enninga, Jost

    2016-01-01

    Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

  15. Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation.

    Science.gov (United States)

    McEwan, William A; Falcon, Benjamin; Vaysburd, Marina; Clift, Dean; Oblak, Adrian L; Ghetti, Bernardino; Goedert, Michel; James, Leo C

    2017-01-17

    Alzheimer's disease (AD) and other neurodegenerative disorders are associated with the cytoplasmic aggregation of microtubule-associated protein tau. Recent evidence supports transcellular transfer of tau misfolding (seeding) as the mechanism of spread within an affected brain, a process reminiscent of viral infection. However, whereas microbial pathogens can be recognized as nonself by immune receptors, misfolded protein assemblies evade detection, as they are host-derived. Here, we show that when misfolded tau assemblies enter the cell, they can be detected and neutralized via a danger response mediated by tau-associated antibodies and the cytosolic Fc receptor tripartite motif protein 21 (TRIM21). We developed fluorescent, morphology-based seeding assays that allow the formation of pathological tau aggregates to be measured in situ within 24 h in the presence of picomolar concentrations of tau seeds. We found that anti-tau antibodies accompany tau seeds into the cell, where they recruit TRIM21 shortly after entry. After binding, TRIM21 neutralizes tau seeds through the activity of the proteasome and the AAA ATPase p97/VCP in a similar manner to infectious viruses. These results establish that intracellular antiviral immunity can be redirected against host-origin endopathogens involved in neurodegeneration.

  16. Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

    Directory of Open Access Journals (Sweden)

    Yu J. Shen

    2015-04-01

    Full Text Available The DNA damage response (DDR induces the expression of type I interferons (IFNs, but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.

  17. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression*

    Science.gov (United States)

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.

    2015-01-01

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. PMID:25623070

  18. RNA polymerase III regulates cytosolic RNA:DNA hybrids and intracellular microRNA expression.

    Science.gov (United States)

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J

    2015-03-20

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Endoplasmic reticulum calcium regulates the retrotranslocation of Trypanosoma cruzi calreticulin to the cytosol.

    Directory of Open Access Journals (Sweden)

    Carlos A Labriola

    2010-10-01

    Full Text Available For most secretory pathway proteins, crossing the endoplasmic reticulum (ER membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD, is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C, since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.

  20. DMPD: Regulation of arachidonic acid release and cytosolic phospholipase A2activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10080535 Regulation of arachidonic acid release and cytosolic phospholipase A2activ...on of arachidonic acid release and cytosolic phospholipase A2activation. PubmedID 10080535 Title Regulation ...of arachidonic acid release and cytosolic phospholipase A2activation. Authors Gij

  1. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression*

    OpenAIRE

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.

    2015-01-01

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cy...

  2. A rat brain cytosolic N-methyltransferase(s) activity converting phosphorylethanolamine into phosphorylcholine.

    Science.gov (United States)

    Andriamampandry, C; Massarelli, R; Freysz, L; Kanfer, J N

    1990-09-14

    It had been previously speculated upon but never proved that the methylation of phosphorylethanolamine could contribute to the production of choline containing compounds. However, experimental evidence obtained with neuronal cultures was interpreted as showing that the stepwise methylation of phosphobases may be an important route for this biosynthesis. We demonstrate that cytosolic fraction from rat brain possesses a N-methyltransferase activity capable of methylating phosphorylethanolamine and its mono- and dimethyl-derivatives into phosphorylcholine. The level of activity detectable in rat liver cytosol is only 18% of that found in the brain cytosol.

  3. Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Marion Le Coadic

    Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

  4. Expression of N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

    Directory of Open Access Journals (Sweden)

    Shuji Mizumoto

    2013-01-01

    Full Text Available Chondroitin sulfate (CS containing E-disaccharide units, glucuronic acid-N-acetylgalactosamine(4, 6-O-disulfate, at surfaces of tumor cells plays a key role in tumor metastasis. However, the molecular mechanism of the metastasis involving the CS chain-containing E-units is not fully understood. In this study, to clarify the role of E-units in the metastasis and to search for potential molecular targets for anticancer drugs, the isolation and characterization of Lewis lung carcinoma (LLC cells stably downregulated by the knockdown for the gene encoding N-acetylgalactosamine 4-O-sulfate 6-O-sulfotransferase (GalNAc4S-6ST, which is responsible for the formation of E-units in CS chains, were performed. Knockdown of GalNAc4S-6ST in LLC cells resulted in a reduction in the proportion of E-units, in adhesiveness to extracellular matrix adhesion molecules and in proliferation in vitro. Furthermore, the stable downregulation of GalNAc4S-6ST expression in LLC cells markedly inhibited the colonization of the lungs by inoculated LLC cells and invasive capacity of LLC cells. These results provide clear evidence that CS chain-containing E-units and/or GalNAc4S-6ST play a crucial role in pulmonary metastasis at least through the increased adhesion and the invasive capacity of LLC cells and also provides insights into future drug targets for anticancer treatment.

  5. Human Cytosolic Extracts Stabilize the HIV-1 Core

    Science.gov (United States)

    Fricke, Thomas; Brandariz-Nuñez, Alberto; Wang, Xiaozhao; Smith, Amos B.

    2013-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects on HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure the stability of in vitro-assembled HIV-1 CA-NC complexes. The assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core. Interestingly, stabilization of in vitro-assembled HIV-1 CA-NC complexes is not due solely to macromolecular crowding, suggesting the presence of specific cellular factors that stabilize the HIV-1 core. By using our novel assay, we measured the abilities of different drugs, such as PF74, CAP-1, IXN-053, cyclosporine, Bi2 (also known as BI-2), and the peptide CAI, to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes. Interestingly, we found that PF74 and Bi2 strongly stabilized HIV-1 CA-NC complexes. On the other hand, the peptide CAI destabilized HIV-1 CA-NC complexes. We also found that purified cyclophilin A destabilizes in vitro-assembled HIV-1 CA-NC complexes in the presence of cellular extracts in a cyclosporine-sensitive manner. In agreement with previous observations using the fate-of-the-capsid assay, we also demonstrated the ability of recombinant CPSF6 to stabilize HIV-1 CA-NC complexes. Overall, our findings suggested that cellular extracts specifically stabilize the HIV-1 core. We believe that our assay can be a powerful tool to assess HIV-1 core stability in vitro. PMID:23885082

  6. Cytosolic superoxide dismutase can provide protection against Fasciola gigantica.

    Science.gov (United States)

    Jaikua, Wipaphorn; Kueakhai, Pornanan; Chaithirayanon, Kulathida; Tanomrat, Rataya; Wongwairot, Sirima; Riengrojpitak, Suda; Sobhon, Prasert; Changklungmoa, Narin

    2016-10-01

    Superoxide dismutases (SOD), antioxidant metallo-enzymes, are a part of the first line of defense in the trematode parasites which act as the chief scavengers for reactive oxygen species (ROS). A recombinant Fasciola gigantica cytosolic SOD (FgSOD) was expressed in Escherichia coli BL21 (DE3) and used for immunizing rabbits to obtain polyclonal antibodies (anti-rFgSOD). This rabbit anti-rFgSOD reacted with the native FgSOD at a molecular weight of 17.5kDa. The FgSOD protein was expressed at high level in parenchyma, caecal epithelium and egg of the parasite. The rFgSOD reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 2, 5, and 7 weeks after infection, and reacted with sera of infected mice. Anti-rFgSOD exhibited cross reactivity with the other parasites' antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. A vaccination was performed in imprinting control region (ICR) mice by subcutaneous injection with 50μg of rFgSOD combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 15 metacercariae by oral route. IgG1 and IgG2a in the immune sera were determined to indicate Th2 and Th1 immune responses. It was found that the parasite burden was reduced by 45%, and both IgG1 and IgG2a levels showed correlation with the numbers of worm recoveries. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Both genome and cytosol dynamics change in E. coli challenged with sublethal rifampicin

    Science.gov (United States)

    Wlodarski, Michal; Raciti, Bianca; Kotar, Jurij; Cosentino Lagomarsino, Marco; Fraser, Gillian M.; Cicuta, Pietro

    2017-02-01

    While the action of many antimicrobial drugs is well understood at the molecular level, a systems-level physiological response to antibiotics remains largely unexplored. This work considers fluctuation dynamics of both the chromosome and cytosol in Escherichia coli, and their response to sublethal treatments of a clinically important antibiotic, rifampicin. We precisely quantify the changes in dynamics of chromosomal loci and cytosolic aggregates (a rheovirus nonstructural protein known as μNS-GFP), measuring short time-scale displacements across several hours of drug exposure. To achieve this we develop an empirical method correcting for photo-bleaching and loci size effects. This procedure allows us to characterize the dynamic response to rifampicin in different growth conditions, including a customised microfluidic device. We find that sub-lethal doses of rifampicin cause a small but consistent increase in motility of both the chromosomal loci and cytosolic aggregates. Chromosomal and cytosolic responses are consistent with each other and between different growth conditions.

  8. Protein abundance profiling of the Escherichia coli cytosol

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    Mann Matthias

    2008-02-01

    Full Text Available Abstract Background Knowledge about the abundance of molecular components is an important prerequisite for building quantitative predictive models of cellular behavior. Proteins are central components of these models, since they carry out most of the fundamental processes in the cell. Thus far, protein concentrations have been difficult to measure on a large scale, but proteomic technologies have now advanced to a stage where this information becomes readily accessible. Results Here, we describe an experimental scheme to maximize the coverage of proteins identified by mass spectrometry of a complex biological sample. Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. A measure of abundance is presented for each of the identified proteins, based on the recently developed emPAI approach which takes into account the number of sequenced peptides per protein. The values of abundance are within a broad range and accurately reflect independently measured copy numbers per cell. As expected, the most abundant proteins were those involved in protein synthesis, most notably ribosomal proteins. Proteins involved in energy metabolism as well as those with binding function were also found in high copy number while proteins annotated with the terms metabolism, transcription, transport, and cellular organization were rare. The barrel-sandwich fold was found to be the structural fold with the highest abundance. Highly abundant proteins are predicted to be less prone to aggregation based on their length, pI values, and occurrence patterns of hydrophobic stretches. We also find that abundant proteins tend to be predominantly essential. Additionally we observe a significant correlation between protein and mRNA abundance in E. coli cells. Conclusion Abundance measurements for more than 1000 E. coli proteins presented in this work

  9. Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit.

    Science.gov (United States)

    Herington, A C; Ymer, S; Roupas, P; Stevenson, J

    1986-04-11

    Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue.

  10. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay.

    Science.gov (United States)

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil; Kim, Dong-Myung; Yoo, Tae Hyeon; Kim, Yong-Sung

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

  12. Down-regulation of N-deacetylase-N-sulfotransferase-1 signaling in the developing diaphragmatic vasculature of nitrofen-induced congenital diaphragmatic hernia.

    Science.gov (United States)

    Takahashi, Toshiaki; Friedmacher, Florian; Zimmer, Julia; Puri, Prem

    2017-06-01

    Congenital diaphragmatic hernia (CDH) has been attributed to various developmental abnormalities of the underlying tissue components. N-deacetylase-N-sulfotransferase-1 (Ndst1) is a strongly expressed biosynthetic enzyme in endothelial cells, which has recently been identified as an important factor during diaphragmatic vascularization. Loss of endothelial Ndst1 has been demonstrated to cause angiogenic defects in the developing diaphragm and disrupt normal diaphragmatic development. Furthermore, deficiency of Ndst1 diminishes the expression of slit homolog 3 (Slit3), a known CDH-related gene that has been associated with reduced vascular density and muscle defects in the diaphragm of Slit3 -/- mice. We hypothesized that expression of Ndst1 and Slit3 is decreased in the diaphragmatic vasculature of fetal rats with nitrofen-induced CDH. Time-mated rats received either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms were microdissected on D13, D15 and D18, and divided into control and nitrofen-exposed specimens. Gene expression levels of Ndst1 and Slit3 were assessed using qRT-PCR. Immunofluorescence-double-staining for Ndst1 and Slit3 was performed to evaluate protein expression and localization. Relative mRNA expression of Ndst1 and Slit3 was significantly decreased in pleuroperitoneal folds (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of nitrofen-exposed fetuses compared to controls. Confocal-laser-scanning-microscopy revealed markedly diminished Ndst1 and Slit3 expression in endothelial cells within the diaphragmatic vasculature on D13, D15 and D18 compared to controls. Down-regulation of Ndst1 signaling in the developing diaphragm may impair endothelial cell migration and angiogenesis, thus leading to defective diaphragmatic vascular development and CDH. Ib. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The fusion of genomes leads to more options: A comparative investigation on the desulfo-glucosinolate sulfotransferases of Brassica napus and homologous proteins of Arabidopsis thaliana.

    Science.gov (United States)

    Hirschmann, Felix; Papenbrock, Jutta

    2015-06-01

    Sulfotransferases (SOTs) (EC 2.8.2.-) play a crucial role in the glucosinolate (Gl) biosynthesis, by catalyzing the final step of the core glucosinolate formation. In Arabidopsis thaliana the three desulfo (ds)-Gl SOTs AtSOT16, AtSOT17 and AtSOT18 were previously characterized, showing different affinities to ds-Gls. But can the knowledge about these SOTs be generally transferred to other Gl-synthesizing plants? It was investigated how many SOTs are present in the economically relevant crop plant Brassica napus L., and if it is possible to predict their characteristics by sequence analysis. The recently sequenced B. napus is a hybrid of Brassica rapa and Brassica oleracea. By database research, 71 putative functional BnSOT family members were identified and at least eleven of those are putative ds-Gl SOTs. Besides the homologs of AtSOT16 - 18, phylogenetic analyses revealed new subfamilies of ds-Gl SOTs, which are not present in A. thaliana. Three of the B. napus ds-Gl SOT proteins were expressed and purified, and characterized by determining the substrate affinities to different ds-Gls. Two of them, BnSOT16-a and BnSOT16-b, showed a significantly higher affinity to an indolic ds-Gl, similarly to AtSOT16. Additionally, BnSOT17-a was characterized and showed a higher affinity to long chained aliphatic Gls, similarly to AtSOT17. Identification of homologs to AtSOT18 was less reliable, because putative SOT18 sequences are more heterogeneous and confirmation of similar characteristics was not possible. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  14. Lack of Tyrosylprotein Sulfotransferase-2 Activity Results in Altered Sperm-Egg Interactions and Loss of ADAM3 and ADAM6 in Epididymal Sperm*

    Science.gov (United States)

    Marcello, Matthew R.; Jia, Weitao; Leary, Julie A.; Moore, Kevin L.; Evans, Janice P.

    2011-01-01

    Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility. PMID:21339297

  15. Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium.

    Directory of Open Access Journals (Sweden)

    Vivek Mishra

    Full Text Available Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825 on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated

  16. Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

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    Helena H. Chowdhury

    2014-10-01

    Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

  17. Measuring Changes in Cytosolic Calcium Levels in HBV- and HBx-Expressing Cultured Primary Hepatocytes.

    Science.gov (United States)

    Casciano, Jessica C; Bouchard, Michael J

    2017-01-01

    Chronic infection with hepatitis B virus (HBV) remains a major worldwide health concern and is the leading cause of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is the only regulatory protein encoded in the HBV genome; HBx stimulates HBV replication in vivo and in vitro. HBx also regulates cytosolic Ca(2+) signaling, and altered Ca(2+) signaling is associated with the development of many diseases, including HCC. Importantly, many HBx functions, including HBx modulation of cell proliferation, apoptosis, and transcription pathways, have been linked to changes in cytosolic Ca(2+) signaling. Additionally, several stages of HBV replication, including capsid formation and activation of the HBV polymerase, are dependent on intracellular Ca(2+). Consequently, defining the molecular mechanism that underlies HBV and HBx modulation of cytosolic Ca(2+) levels is important for understanding HBV pathogenesis and the role of HBx in HBV replication. Here, we describe a single-cell Ca(2+)-imaging protocol that we use to investigate HBV and HBx effects on the level of cytosolic Ca(2+). We specifically outline two methods that we use to evaluate HBV and HBx regulation of cytosolic Ca(2+) levels in cultured primary hepatocytes. This protocol can also be adapted for use in liver cell lines.

  18. Micrometer-Scale Membrane Transition of Supported Lipid Bilayer Membrane Reconstituted with Cytosol of Dictyostelium discoideum

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    Kei Takahashi

    2017-03-01

    Full Text Available Background: The transformation of the supported lipid bilayer (SLB membrane by extracted cytosol from living resources, has recently drawn much attention. It enables us to address the question of whether the purified phospholipid SLB membrane, including lipids related to amoeba locomotion, which was discussed in many previous studies, exhibits membrane deformation in the presence of cytosol extracted from amoeba; Methods: In this report, a method for reconstituting a supported lipid bilayer (SLB membrane, composed of purified phospholipids and cytosol extracted from Dictyostelium discoideum, is described. This technique is a new reconstitution method combining the artificial constitution of membranes with the reconstitution using animate cytosol (without precise purification at a molecular level, contributing to membrane deformation analysis; Results: The morphology transition of a SLB membrane composed of phosphatidylcholines, after the addition of cytosolic extract, was traced using a confocal laser scanning fluorescence microscope. As a result, pore formation in the SLB membrane was observed and phosphatidylinositides incorporated into the SLB membrane tended to suppress pore formation and expansion; Conclusions: The current findings imply that phosphatidylinositides have the potential to control cytoplasm activity and bind to a phosphoinositide-containing SLB membrane.

  19. An assay for clogging the ciliary pore complex distinguishes mechanisms of cytosolic and membrane protein entry.

    Science.gov (United States)

    Takao, Daisuke; Dishinger, John F; Kee, H Lynn; Pinskey, Justine M; Allen, Ben L; Verhey, Kristen J

    2014-10-06

    As a cellular organelle, the cilium contains a unique protein composition. Entry of both membrane and cytosolic components is tightly regulated by gating mechanisms at the cilium base; however, the mechanistic details of ciliary gating are largely unknown. We previously proposed that entry of cytosolic components is regulated by mechanisms similar to those of nuclear transport and is dependent on nucleoporins (NUPs), which comprise a ciliary pore complex (CPC). To investigate ciliary gating mechanisms, we developed a system to clog the pore by inhibiting NUP function via forced dimerization. We targeted NUP62, a component of the central channel of the nuclear pore complex (NPC), for forced dimerization by tagging it with the homodimerizing Fv domain. As proof of principle, we show that forced dimerization of NUP62-Fv attenuated (1) active transport of BSA into the nuclear compartment and (2) the kinesin-2 motor KIF17 into the ciliary compartment. Using the pore-clogging technique, we find that forced dimerization of NUP62 attenuated the gated entry of cytosolic proteins but did not affect entry of membrane proteins or diffusional entry of small cytosolic proteins. We propose a model in which active transport of cytosolic proteins into both nuclear and ciliary compartments requires functional NUPs of the central pore, whereas lateral entry of membrane proteins utilizes a different mechanism that is likely specific to each organelle's limiting membrane. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Cytosolic and membrane-bound chitinases of two mucoraceous fungi: a comparative study.

    Science.gov (United States)

    Balasubramanian, R; Manocha, M S

    1992-04-01

    Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS-PAGE the chitinase from both fungi migrated as a single band of M(r) 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS-PAGE showed two bands for each fungus with M(r) of 69.5 and 55 kDa in C. cucurbitarum and M(r) 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N'-diacetylchitobiose, and N,N',N"-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but beta-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar.

  1. ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

    KAUST Repository

    Houben, Diane

    2012-05-08

    Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

  2. The Cytosolic pH of Individual Saccharomyces cerevisiae Cells Is a Key Factor in Acetic Acid Tolerance

    Science.gov (United States)

    Fernández-Niño, Miguel; Marquina, Maribel; Swinnen, Steve; Rodríguez-Porrata, Boris

    2015-01-01

    It was shown recently that individual cells of an isogenic Saccharomyces cerevisiae population show variability in acetic acid tolerance, and this variability affects the quantitative manifestation of the trait at the population level. In the current study, we investigated whether cell-to-cell variability in acetic acid tolerance could be explained by the observed differences in the cytosolic pHs of individual cells immediately before exposure to the acid. Results obtained with cells of the strain CEN.PK113-7D in synthetic medium containing 96 mM acetic acid (pH 4.5) showed a direct correlation between the initial cytosolic pH and the cytosolic pH drop after exposure to the acid. Moreover, only cells with a low initial cytosolic pH, which experienced a less severe drop in cytosolic pH, were able to proliferate. A similar correlation between initial cytosolic pH and cytosolic pH drop was also observed in the more acid-tolerant strain MUCL 11987-9. Interestingly, a fraction of cells in the MUCL 11987-9 population showed initial cytosolic pH values below the minimal cytosolic pH detected in cells of the strain CEN.PK113-7D; consequently, these cells experienced less severe drops in cytosolic pH. Although this might explain in part the difference between the two strains with regard to the number of cells that resumed proliferation, it was observed that all cells from strain MUCL 11987-9 were able to proliferate, independently of their initial cytosolic pH. Therefore, other factors must also be involved in the greater ability of MUCL 11987-9 cells to endure strong drops in cytosolic pH. PMID:26341199

  3. Isolation and characterization from porcine serum of a soluble sulfotransferase responsible for 6-O-sulfation of the galactose residue in 2'-fucosyllactose: implications in the synthesis of the ligand for L-selectin.

    Science.gov (United States)

    Huynh, Q K; Shailubhai, K; Boddupalli, H; Yu, H H; Broschat, K O; Jacob, G S

    1999-07-01

    A soluble sulfotransferase from porcine serum which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) to 2'-fucosyllactose (2'-FL) was purified 36,333-fold using a combination of conventional and affinity chromatographic steps. The purified enzyme preparation after non-denaturing discontinuous-PAGE exhibited a molecular mass of about 80 kDa by reducing SDS-PAGE. However, when a partially purified enzyme preparation was subjected to gel filtration on Sephacryl S-300, the enzyme activity eluted in the void volume, which indicated that the native enzyme existed as an oligomer. The purified enzyme showed Km values of 9.15 microM for PAPS and 15.38 mM for 2'-FL at the optimum pH value of 7.4. The substrate specificity of the purified enzyme was evaluated with various sugars that are structurally similar to sialyl LewisX (sLeX). Results indicated that 3'-sialyllactose and lactose were efficient acceptors of sulfation, whereas 6'-sialyllactose and 6'-sialyllactosamine were poor substrates for this sulfotransferase. Further, the reaction product analysis revealed that the sulfate substitution, when using 2'-FL as the substrate, was at the C-6 position of the galactose residue. Coincidentally, a similar enzyme activity was also found in porcine lymphoid tissues such as, lymph nodes (peripheral and mesenteric) and spleen. Collectively, these findings suggest that this enzyme might be involved in the synthesis of the ligand for L-selectin.

  4. Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

    Science.gov (United States)

    Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

    2010-01-01

    Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

  5. Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

    Science.gov (United States)

    Böldicke, Thomas

    2017-05-01

    Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic. © 2017 The Protein Society.

  6. Curcuminoids inhibit multiple human cytochromes P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes, while piperine is a relatively selective CYP3A4 inhibitor

    Science.gov (United States)

    Volak, Laurie P.; Ghirmai, Senait; Cashman, John R.; Court, Michael H.

    2008-01-01

    Curcuminoid extract and piperine are being evaluated for beneficial effects in Alzheimer’s disease, among other intractable disorders. Consequently, we studied the potential for herb-drug interactions involving cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes. The curcuminoid extract inhibited SULT > CYP2C19 > CYP2B6 > UGT > CYP2C9 > CYP3A activities with IC50 values ranging from 0.99 ± 0.04 to 25.3 ± 1.3 μM, while CYP2D6, CYP1A2, and CYP2E1 activities were less affected (IC50 values >60 μM). Inhibition of CYP3A activity by curcuminoid extract was consistent with competitive inhibition (Ki = 11.0 ± 1.3 μM), while inhibition of both CYP2C9 and CYP2C19 activities were consistent with mixed competitive-noncompetitive inhibition (10.6 ± 1.1 μM and 7.8 ± 0.9 μM, respectively). Piperine was a relatively selective noncompetitive inhibitor of CYP3A (IC50 5.5 ± 0.7 μM, Ki = 5.4 ± 0.3 μM) with less effect on other enzymes evaluated (IC50 >29 μM). Curcuminoid extract and piperine inhibited recombinant CYP3A4 much more potently (by >5-fold) than CYP3A5. Pure synthetic curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) were also evaluated for their effects on CYP3A, CYP2C9, UGT, and SULT activities. All three curcuminoids had similar effects on CYP3A, UGT, and SULT activity, but demethoxycurcumin (IC50 = 8.8 ± 1.2 μM) was more active against CYP2C9 than either curcumin or bisdemethoxycurcumin (IC50 >50 μM). Based on these data and expected tissue concentrations of inhibitors, we predict that an orally administered curcuminoid/piperine combination is most likely to inhibit CYP3A, CYP2C9, UGT, and SULT metabolism within the intestinal mucosa. PMID:18480186

  7. Chlorinated Biphenyl Quinones and Phenyl-2,5-benzoquinone Differentially Modify the Catalytic Activity of Human Hydroxysteroid Sulfotransferase hSULT2A1

    Science.gov (United States)

    Qin, Xiaoyan; Lehmler, Hans-Joachim; Teesch, Lynn M.; Robertson, Larry W.; Duffel, Michael W.

    2013-01-01

    Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of environmental chemicals, drugs, and other xenobiotics in addition to endogenous compounds that include hydroxysteroids and bile acids. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and oxidized metabolites of PCBs may play significant roles in the etiology of their adverse health effects. Quinones derived from oxidative metabolism of PCBs (PCB-quinones) react with nucleophilic sites in proteins and also undergo redox cycling to generate reactive oxygen species. This, along with the sensitivity of hSULT2A1 to oxidative modification at cysteine residues led us to hypothesize that electrophilic PCB-quinones react with hSULT2A1 to alter its catalytic function. Thus, we examined the effects of four phenylbenzoquinones on the ability of hSULT2A1 to catalyze the sulfation of the endogenous substrate, dehydroepiandrosterone (DHEA). The quinones studied were 2′-chlorophenyl-2,5-benzoquinone (2′-Cl-BQ), 4′-chlorophenyl-2,5-benzoquinone (4′-Cl-BQ), 4′-chlorophenyl-3,6-dichloro-2,5-benzoquinone (3,6,4′-triCl-BQ), and phenyl-2,5-benzoquinone (PBQ). At all concentrations examined, pretreatment of hSULT2A1 with the PCB-quinones decreased catalytic activity of hSULT2A1. Pretreatment with low concentrations of PBQ, however, increased the catalytic activity of the enzyme, while higher concentrations inhibited catalysis. A decrease in substrate inhibition with DHEA was seen following preincubation of hSULT2A1 with all of the quinones. Proteolytic digestion of the enzyme followed by LC/MS analysis indicated PCB-quinone- and PBQ-adducts at Cys55 and Cys199, as well as oxidation products at methionines in the protein. Equilibrium binding experiments and molecular modeling suggested that changes due to these modifications may affect the nucleotide binding site and the entrance to the sulfuryl acceptor binding site of hSULT2A1. PMID:24059442

  8. Structure and role of neutrophil cytosol factor 1 (NCF1) gene in ...

    African Journals Online (AJOL)

    Yomi

    2010-12-27

    Dec 27, 2010 ... in innate immunity and produce reactive oxygen species and reduce the severity and duration of parasitic infection and autoimmune disease. NCF1 also has a role in T cell activation. Key words: Neutrophil cytosol factor 1 (NCF1) gene, exons, T cell activation. INTRODUCTION. An immune system is a ...

  9. Changes in the level of cytosolic calcium, nitric oxide and nitric oxide ...

    Indian Academy of Sciences (India)

    Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis. In the present investigation, we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium ...

  10. Enzymatic Regulation of Cytosolic Thymidine Kinase 1 and Mitochondrial Thymidine Kinase 2

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte

    2010-01-01

    . Apart from the complex de novo synthesis of dTTP through UDP reduction, dTTP is provided through salvage of thymidine catalyzed by the thymidine kinases, the cytosolic and cell cycle regulated TK1 and the mitochondrial and constitutively expressed TK2. The complex enzymatic regulation of TK1 and TK2...

  11. Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver.

    Science.gov (United States)

    Hu, M L; Viljoen, A J; Tappel, A L

    1988-04-01

    Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.

  12. A reporter of UV intensity delivered to the cytosol during photolytic uncaging

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Dewitt, Sharon; Hallett, Maurice B

    2010-01-01

    Photolytic uncaging of biologically-active molecules within cells is a powerful technique. However, the delivery of uncaging light into the cytosol can vary between cell types, individual cells of the same type, and different loci within an individual cell because of optical differences in absorb...

  13. Cytosolic beta-glycosidases for activation of glycoside prodrugs of daunorubicin

    NARCIS (Netherlands)

    Pinedo, H.M.; Quadir, R.; Haisma, H.J.; Boven, E.

    2003-01-01

    Human cytosolic beta-glycosidase is a small monomeric enzyme that is active under physiological conditions, which might be ideal for enzyme-prodrug therapy. We have previously reported the synthesis of a galactoside (DNR-GlA(3)) and a glucoside (DNR-GsA3) prodrug of daunorubicin. In the present

  14. Structure and role of neutrophil cytosol factor 1 (NCF1) gene in ...

    African Journals Online (AJOL)

    Yomi

    2010-12-27

    Dec 27, 2010 ... The neutrophil cytosol factor 1 (NCF1) gene consists of 11 exons and is found in two forms; one is wild type gene and the other is pseudogene. It has more than 98% homology. Both genes occupy the same chromosome region. The mutation in this gene leads to various types of diseases such as chronic.

  15. The cytosolic B-glucosidase GBA3 does not influence type 1 Gaucher disease manifestation

    NARCIS (Netherlands)

    Dekker, N.; Voorn-Brouwer, T.; Verhoek, M.; Wennekes, T.; Narayan, R.S.; Speijer, D.; Hollak, C.E.M.; Overkleeft, H.S.; Boot, R.G.; Aerts, J.M.F.G.

    2011-01-01

    GBA3, also known as cytosolic ß-glucosidase, is thought to hydrolyze xenobiotic glycosides in man. Deficiency of glucocerebrosidase (GBA), a ß-glucosidase degrading glucosylceramide, underlies Gaucher disease. We examined GBA3, which recently was proposed to degrade glucosylceramide and influence

  16. The cytosolic beta-glucosidase GBA3 does not influence type 1 Gaucher disease manifestation

    NARCIS (Netherlands)

    Dekker, Nick; Voorn-Brouwer, Tineke; Verhoek, Marri; Wennekes, Tom; Narayan, Ravi S.; Speijer, Dave; Hollak, Carla E. M.; Overkleeft, Hermen S.; Boot, Rolf G.; Aerts, Johannes M. F. G.

    2011-01-01

    GBA3, also known as cytosolic beta-glucosidase, is thought to hydrolyze xenobiotic glycosides in man. Deficiency of glucocerebrosidase (GBA), a beta-glucosidase degrading glucosylteramide, underlies Gaucher disease. We examined GBA3, which recently was proposed to degrade glucosylceramide and

  17. Assay of oestrogen and progestin receptors in human meningioma cytosols using immunological methods

    NARCIS (Netherlands)

    Blankenstein, M.A.; Meulen-Dijk, C. van der; Thijssen, J.H.H.

    1987-01-01

    Oestrogen (ER) and progestin receptors (PR) were assayed in human meningioma cytosol by radioligand binding assay with Scatchard plot analysis and by monoclonal antibody based enzyme immunoassays. For comparison, human breast cancer tissues were used. Results of both assays agreed very well. For

  18. Live cell imaging of cytosolic NADH/NAD+ratio in hepatocytes and liver slices.

    Science.gov (United States)

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

  19. Diversity of cytosolic HSP70 Heat Shock Protein from decapods and their phylogenetic placement within Arthropoda.

    Science.gov (United States)

    Baringou, Stephane; Rouault, Jacques-Deric; Koken, Marcel; Hardivillier, Yann; Hurtado, Luis; Leignel, Vincent

    2016-10-10

    The 70kDa heat shock proteins (HSP70) are considered the most conserved members of the HSP family. These proteins are primordial to the cell, because of their implications in many cellular pathways (e. g., development, immunity) and also because they minimize the effects of multiple stresses (e. g., temperature, pollutants, salinity, radiations). In the cytosol, two ubiquitous HSP70s with either a constitutive (HSC70) or an inducible (HSP70) expression pattern are found in all metazoan species, encoded by 5 or 6 genes (Drosophila melanogaster or yeast and human respectively). The cytosolic HSP70 protein family is considered a major actor in environmental adaptation, and widely used in ecology as an important biomarker of environmental stress. Nevertheless, the diversity of cytosolic HSP70 remains unclear amongst the Athropoda phylum, especially within decapods. Using 122 new and 311 available sequences, we carried out analyses of the overall cytosolic HSP70 diversity in arthropods (with a focus on decapods) and inferred molecular phylogenies. Overall structural and phylogenetic analyses showed a surprisingly high diversity in cytosolic HSP70 and revealed the existence of several unrecognised groups. All crustacean HSP70 sequences present signature motifs and molecular weights characteristic of non-organellar HSP70, with multiple specific substitutions in the protein sequence. The cytosolic HSP70 family in arthropods appears to be constituted of at least three distinct groups (annotated as A, B and C), which comprise several subdivisions, including both constitutive and inducible forms. Group A is constituted by several classes of Arthropods, while group B and C seem to be specific to Malacostraca and Hexapoda/Chelicerata, respectively. The HSP70 organization appeared much more complex than previously suggested, and far beyond a simple differentiation according to their expression pattern (HSC70 versus HSP70). This study proposes a new classification of cytosolic

  20. Reduction of dimethylarsinic acid to the highly toxic dimethylarsinous acid by rats and rat liver cytosol.

    Science.gov (United States)

    Németi, Balázs; Gregus, Zoltán

    2013-03-18

    Dimethylarsinic acid (DMAs(V)), the major urinary metabolite of inorganic arsenic, is weakly cytotoxic, whereas its reduced form, dimethylarsinous acid (DMAs(III)), is highly toxic. Although glutathione S-transferase omega 1 (GSTO1) and arsenic methyltransferase have been shown or thought to catalyze DMAs(V) reduction, their role in DMAs(V) reduction in vivo, or in cell extracts is uncertain. Therefore, the reduction of DMAs(V) to DMAs(III) in rats and in rat liver cytosol was studied to better understand its mechanism. To assess DMAs(V) reduction in rats, a novel procedure was devised based on following the accumulation of red blood cell (RBC)-bound dimethylarsenic (DMAs), which represents DMAs(III), in the blood of DMAs(V)-injected anesthetized rats. These studies indicated that rats reduced DMAs(V) to DMAs(III) to a significant extent, as in 90 min 31% of the injected 50 μmol/kg DMAs(V) dose was converted to DMAs(III) that was sequestered by the circulating erythrocytes. Pretreatment of rats with glutathione (GSH) depletors (phorone or BSO) delayed the elimination of DMAs(V) and the accumulation of RBC-bound DMAs, whereas the indirect methyltransferase inhibitor periodate-oxidized adenosine was without effect. Assessment of DMAs(V)-reducing activity of rat liver cytosol revealed that reduction of DMAs(V) required cytosolic protein and GSH and was inhibited by thiol reagents, GSSG and dehydroascorbate. Although thioredoxin reductase (TRR) inhibitors (aurothioglucose and Sb(III)) inhibited cytosolic DMAs(V) reduction, recombinant rat TRR plus NADPH, alone or when added to the cytosol, failed to support DMAs(V) reduction. On ultrafiltration of the cytosol through a 3 kDa filter, the reducing activity in the retentate was lost but was largely restored by NADPH. Such experiments also suggested that the reducing enzyme was larger than 100 kDa and was not GSTO1. In summary, reduction of DMAs(V) to the highly toxic DMAs(III) in rats and rat liver cytosol is a GSH

  1. Formation of disulfide bridges by a single-chain Fv antibody in the reducing ectopic environment of the plant cytosol

    NARCIS (Netherlands)

    Schouten, A.; Roosien, J.; Bakker, J.; Schots, A.

    2002-01-01

    Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event and is mostly linked to inactivation of protein activity. In this report the in vivo redox state of a single-chain Fv (scFv) antibody fragment in the plant cytosol was investigated. The scFv antibody

  2. Mitochondrial Iron-Sulfur Cluster Activity and Cytosolic Iron Regulate Iron Traffic in Saccharomyces cerevisiae.

    Science.gov (United States)

    Wofford, Joshua D; Lindahl, Paul A

    2015-11-06

    An ordinary differential equation-based mathematical model was developed to describe trafficking and regulation of iron in growing fermenting budding yeast. Accordingly, environmental iron enters the cytosol and moves into mitochondria and vacuoles. Dilution caused by increasing cell volume is included. Four sites are regulated, including those in which iron is imported into the cytosol, mitochondria, and vacuoles, and the site at which vacuolar Fe(II) is oxidized to Fe(III). The objective of this study was to determine whether cytosolic iron (Fecyt) and/or a putative sulfur-based product of iron-sulfur cluster (ISC) activity was/were being sensed in regulation. The model assumes that the matrix of healthy mitochondria is anaerobic, and that in ISC mutants, O2 diffuses into the matrix where it reacts with nonheme high spin Fe(II) ions, oxidizing them to nanoparticles and generating reactive oxygen species. This reactivity causes a further decline in ISC/heme biosynthesis, which ultimately gives rise to the diseased state. The ordinary differential equations that define this model were numerically integrated, and concentrations of each component were plotted versus the concentration of iron in the growth medium and versus the rate of ISC/heme biosynthesis. Model parameters were optimized by fitting simulations to literature data. The model variant that assumed that both Fecyt and ISC biosynthesis activity were sensed in regulation mimicked observed behavior best. Such "dual sensing" probably arises in real cells because regulation involves assembly of an ISC on a cytosolic protein using Fecyt and a sulfur species generated in mitochondria during ISC biosynthesis and exported into the cytosol. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development.

    Science.gov (United States)

    Pasternak, Maciej; Lim, Benson; Wirtz, Markus; Hell, Rüdiger; Cobbett, Christopher S; Meyer, Andreas J

    2008-03-01

    Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, gamma-glutamate cysteine ligase (GSH1), responsible for synthesis of gamma-glutamylcysteine (gamma-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by gamma-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of gamma-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by gamma-EC in vitro strongly suggests export of gamma-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, gamma-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1.

  4. Limits to anaerobic energy and cytosolic concentration in the living cell

    Science.gov (United States)

    Paglietti, A.

    2015-11-01

    For many physical systems at any given temperature, the set of all states where the system's free energy reaches its largest value can be determined from the system's constitutive equations of internal energy and entropy, once a state of that set is known. Such an approach is fraught with complications when applied to a living cell, because the cell's cytosol contains thousands of solutes, and thus thousands of state variables, which makes determination of its state impractical. We show here that, when looking for the maximum energy that the cytosol can store and release, detailed information on cytosol composition is redundant. Compatibility with cell's life requires that a single variable that represents the overall concentration of cytosol solutes must fall between defined limits, which can be determined by dehydrating and overhydrating the cell to its maximum capacity. The same limits are shown to determine, in particular, the maximum amount of free energy that a cell can supply in fast anaerobic processes, starting from any given initial state. For a typical skeletal muscle in normal physiological conditions this energy, i.e., the maximum anaerobic capacity to do work, is calculated to be about 960 J per kg of muscular mass. Such energy decreases as the overall concentration of solutes in the cytosol is increased. Similar results apply to any kind of cell. They provide an essential tool to understand and control the macroscopic response of single cells and multicellular cellular tissues alike. The applications include sport physiology, cell aging, disease produced cell damage, drug absorption capacity, to mention the most obvious ones.

  5. Oil and protein accumulation in developing seeds is influenced by the expression of a cytosolic pyrophosphatase in Arabidopsis.

    Science.gov (United States)

    Meyer, Knut; Stecca, Kevin L; Ewell-Hicks, Kim; Allen, Stephen M; Everard, John D

    2012-07-01

    This study describes a dominant low-seed-oil mutant (lo15571) of Arabidopsis (Arabidopsis thaliana) generated by enhancer tagging. Compositional analysis of developing siliques and mature seeds indicated reduced conversion of photoassimilates to oil. Immunoblot analysis revealed increased levels of At1g01050 protein in developing siliques of lo15571. At1g01050 encodes a soluble, cytosolic pyrophosphatase and is one of five closely related genes that share predicted cytosolic localization and at least 70% amino acid sequence identity. Expression of At1g01050 using a seed-preferred promoter recreated most features of the lo15571 seed phenotype, including low seed oil content and increased levels of transient starch and soluble sugars in developing siliques. Seed-preferred RNA interference-mediated silencing of At1g01050 and At3g53620, a second cytosolic pyrophosphatase gene that shows expression during seed filling, led to a heritable oil increase of 1% to 4%, mostly at the expense of seed storage protein. These results are consistent with a scenario in which the rate of mobilization of sucrose, for precursor supply of seed storage lipid biosynthesis by cytosolic glycolysis, is strongly influenced by the expression of endogenous pyrophosphatase enzymes. This emphasizes the central role of pyrophosphate-dependent reactions supporting cytosolic glycolysis during seed maturation when ATP supply is low, presumably due to hypoxic conditions. This route is the major route providing precursors for seed oil biosynthesis. ATP-dependent reactions at the entry point of glycolysis in the cytosol or plastid cannot fully compensate for the loss of oil content observed in transgenic events with increased expression of cytosolic pyrophosphatase enzyme in the cytosol. These findings shed new light on the dynamic properties of cytosolic pyrophosphate pools in developing seed and their influence on carbon partitioning during seed filling. Finally, our work uniquely demonstrates that

  6. The Role of RaxST, a Prokaryotic Sulfotransferase, and RaxABC, a Putative Type I Secretion System, in Activation of the Rice XA21-Mediated Immune Response

    Directory of Open Access Journals (Sweden)

    Pamela C. Ronald

    2014-01-01

    Full Text Available Tyrosine sulfation is an important posttranslational modification that determines the outcome of serious diseases in plants and animals. We have recently demonstrated that the plant pathogen Xanthomonas oryzae pv. oryzae (Xoo carries a functional sulfotransferase (RaxST. raxST is required for activation of rice Xa21-mediated immunity indicating the critical, but unknown, function of raxST in mediating the Xoo/rice interaction. The raxST gene resides in the same operon (raxSTAB as components of a predicted type I secretion and processing system (RaxA and RaxB. These observations suggest a model where RaxST sulfates a molecule that contains a leader peptide, which is cleaved by the peptidase domain of the RaxB protein and secreted outside the bacterial cell by the RaxABC T1SS.

  7. Cytosolic Cl- Affects the Anticancer Activity of Paclitaxel in the Gastric Cancer Cell Line, MKN28 Cell

    Directory of Open Access Journals (Sweden)

    Sachie Tanaka

    2017-05-01

    Full Text Available Background/Aims: Our previous study revealed that cytosolic Cl- affected neurite elongation promoted via assembly of microtubule in rat pheochromocytoma PC12D cells and Cl-–induced blockade of intrinsic GTPase enhanced tubulin polymerization in vitro. Paclitaxel (PTX is a microtubule-targeted chemotherapeutic drug and stabilizes microtubules resulting in mainly blockade of mitosis at the metaphase-anaphase transition and induction of apoptosis. In the present study, we tried to clarify whether the cytosolic Cl- affected PTX ability to inhibit cell growth in the gastric cancer cell line, MKN28. Methods: To clarify the cytosolic Cl- action on PTX-induced cell death and metaphase-anaphase transition in the gastric cancer cell line, MKN28 cell, and PTX-induced tubulin polymerization, we performed cell proliferation assay, cytosolic Cl- concentration measurement, immunofluorescence microscopy, and in vitro tubulin polymerization assay. Results: The decline of cytosolic Cl- weakened the cytotoxic effect of PTX on cell proliferation of MKN28 cells, which could pass through the metaphase-anaphase transition. Moreover, in vitro PTX-induced tubulin polymerization was diminished under the low Cl- condition. Conclusions: Our results strongly suggest that the upregulation of cytosolic Cl- concentration would enhance the antitumor effect of PTX, and that the cytosolic Cl- would be one of the key targets for anti-cancer therapy.

  8. A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

    Directory of Open Access Journals (Sweden)

    Julie Adam

    2013-05-01

    Full Text Available The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH, predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

  9. Neonicotinoid insecticides: reduction and cleavage of imidacloprid nitroimine substituent by liver microsomal and cytosolic enzymes.

    Science.gov (United States)

    Schulz-Jander, Daniel A; Leimkuehler, William M; Casida, John E

    2002-09-01

    The major insecticide imidacloprid (IMI) is known to be metabolized by human cytochrome P450 3A4 with NADPH by imidazolidine hydroxylation and dehydrogenation to give 5-hydroxy-imidacloprid and the olefin, respectively, and by nitroimine reduction and cleavage to yield the nitrosoimine, guanidine, and urea derivatives. More extensive metabolism by human or rabbit liver microsomes with NADPH or rabbit liver cytosol without added cofactor reduces the IMI N-nitro group to an N-amino substituent, i.e., the corresponding hydrazone. A major metabolite on incubation of IMI in the human microsome-NADPH system is tentatively assigned by LC/MS as a 1,2,4-triazol-3-one derived from the hydrazone; the same product is obtained on reaction of the hydrazone with ethyl chloroformate. The hydrazone and proposed triazolone are considered here together (referred to as the hydrazone) for quantitation. Only a portion of the microsomal reduction and cleavage of the nitroimine substituent is attributable to a CYP450 enzyme. The cytosolic enzyme conversion to the hydrazone is inhibited by added cofactors (NAD > NADH > NADP > NADPH) and enhanced by an argon instead of an air atmosphere. The responsible cytosolic enzyme(s) does not appear to be DT-diaphorase (which is inhibited by several neonicotinoids), aldose reductase, aldehyde reductase, or xanthine oxidase. However, the cytosolic metabolism of IMI is inhibited by several aldo-keto-reductase inhibitors (i.e., alrestatin, EBPC, Ponalrestat, phenobarbital, and quercetin). Other neonicotinoids with nitroimine, nitrosoimine, and nitromethylene substituents are probably also metabolized by "neonicotinoid nitro reductase(s)" since they serve as competitive substrates for [(3)H]IMI metabolism.

  10. [Utilization of nitrate by bacteroids and cytosol of nodules formed by Rhizobium leguminosarum].

    Science.gov (United States)

    Fernández-López, M; Delgado, M J; Olivares, J; Bedmar, E J

    1989-06-01

    Nitrite production by nodules and roots of pea plants (Pisum sativum L., cultivar Alaska) inoculated with Rhizobium leguminosarum strain 3855 has been studied. Nitrate reductase (NR) activity and nitrite reductase (NiR) activity of the bacteroidal and cytosolic fractions of the nodules were also determined, as well as the nitrite content of the nodules cytosol. Nitrite production by nodules and roots from plants treated with 5 mM KNO3 was higher than that of nodules and roots from plants not treated with nitrate, and regardless of the nitrate treatment, nitrite production increased with the incubation period. The presence of nitrate, propanol or both compounds in the incubation mixtures significantly increased the nitrite production by nodules and roots. Nitrite reductase activity was detected in fresh by isolated bacteroids of R. leguminosarum strain 3855, although the presence of nitrate reductase activity could not be detected both in bacteroids of nodules isolated from plants treated or not with 5 mM KNO3. After isolation, when bacteroids were incubated in a mixture with nitrate, nitrate reductase activity developed after incubation for 12 h. Consequently, there was an increase in nitrite reductase activity, which resulted in the disappearance of the nitrite previously accumulated in the incubation medium. Nitrate utilization by bacteroids was not detected until 5 h from the beginning of the incubation period. Since the presence of chloramphenicol or rifampicin in the incubation medium prevented the development of the nitrate reductase activity, such activity was induced in bacteroids. Nitrite content and nitrate reductase and nitrite reductase activities of the cytosol from nodules of pea plants treated or not with 5 mM KNO3 varied with the buffer used for nodules homogenization. However, no nitrite was found when nodules were homogenized with ethanol, what indicates that nitrite accumulation in the cytosol occurs during the homogenization process of the

  11. High effective cytosolic H+ buffering in mouse cortical astrocytes attributable to fast bicarbonate transport.

    Science.gov (United States)

    Theparambil, Shefeeq M; Deitmer, Joachim W

    2015-09-01

    Cytosolic H(+) buffering plays a major role for shaping intracellular H(+) shifts and hence for the availability of H(+) for biochemical reactions and acid/base-coupled transport processes. H(+) buffering is one of the prime means to protect the cell from large acid/base shifts. We have used the H(+) indicator dye BCECF and confocal microscopy to monitor the cytosolic H(+) concentration, [H(+)]i, in cultured cortical astrocytes of wild-type mice and of mice deficient in sodium/bicarbonate cotransporter NBCe1 (NBCe1-KO) or in carbonic anhydrase isoform II (CAII-KO). The steady-state buffer strength was calculated from the amplitude of [H(+)]i transients as evoked by CO2/HCO3(-) and by butyric acid in the presence and absence of CO2/HCO3(-). We tested the hypotheses if, in addition to instantaneous physicochemical H(+) buffering, rapid acid/base transport across the cell membrane contributes to the total, "effective" cytosolic H(+) buffering. In the presence of 5% CO2/26 mM HCO3(-), H(+) buffer strength in astrocytes was increased 4-6 fold, as compared with that in non-bicarbonate, HEPES-buffered solution, which was largely attributable to fast HCO3 (-) transport into the cells via NBCe1, supported by CAII activity. Our results show that within the time frame of determining physiological H(+) buffering in cells, fast transport and equilibration of CO2/H(+)/HCO3(-) can make a major contribution to the total "effective" H(+) buffer strength. Thus, "effective" cellular H(+) buffering is, to a large extent, attributable to membrane transport of base equivalents rather than a purely passive physicochemical process, and can be much larger than reported so far. Not only physicochemical H(+) buffering, but also rapid import of HCO3(-) via the electrogenic sodium-bicarbonate cotransporter NBCe1, supported by carbonic anhydrase II (CA II), was identified to enhance cytosolic H(+) buffer strength substantially. © 2015 Wiley Periodicals, Inc.

  12. Unique hepatic cytosolic arginase evolved independently in ureogenic freshwater air-breathing teleost, Heteropneustes fossilis.

    Directory of Open Access Journals (Sweden)

    Shilpee Srivastava

    Full Text Available Hepatic cytosolic arginase (ARG I, an enzyme of the urea cycle operating in the liver of ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55°C. The Km of purified enzyme for L-arginine was 2.65±0.39 mM. L-ornithine and N(ω-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.52±0.02mM and 0.08±0.006mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150-200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of ureotelic vertebrates.

  13. The presence of a protein activator of sarcolemmal polyphosphoinositide phospholipase C in cardiac cytosol

    Energy Technology Data Exchange (ETDEWEB)

    Quist, E.E.; Kriewaldt, S.D.; Powell, P.B. (Texas College of Osteopathic Medicine, Fort Worth (USA))

    1989-01-01

    To study polyphosphoinositide phospholipase (PL) C, isolated sarcolemmal membranes were preincubated with Mg({sup 32}P)-ATP to label phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-diphosphate (PIP{sub 2}). After washing, PLC activity was determined by measuring the release of {sup 32}P-labeled inositol diphosphate (IP{sub 2}) and/or inositol trisphospate (IP{sub 3}) from membrane PIP and PIP{sub 2} during incubation at 25{degree}C and pH 7.4. Increasing concentrations of Ca{sup 2+} (0-100 {mu}M) increased IP{sub 2} by 100% over the 0 Ca{sup 2+} control levels. Ca{sup 2+} dependent PLC hydrolyzed both PIP and PIP{sub 2} with apparent D{sub A}'s of approximately 0.5 and 70 {mu}M. Addition of dialyzed cytosol further increased IP{sub 2} release by 250% without affecting the K{sub A}'s for Ca{sup 2+} activation. The cytosolic activator was partially purified by DEAE Sephacel chromatography was heat labile and sensitive to trypsin pretreatment identifying it as a protein. In contrast, 10 mM NaF increased the Ca{sup 2+} affinity for PLC 2-fold. These results show that cardiac sarcolemma possess a membrane bound Ca{sup 2+} dependent PLC activity which is regulated by a cytosolic protein activator and a G protein. The cytosolic activator would potentially amplify the amount of sarcolemmal polyphosphoinositides hydrolyzed by PLC in response to muscarinic receptor activation by acetylcholine. In addition, activation of PLC by NaF or other G protein activators could result from increasing the Ca{sup 2+} affinity of PLC to physiological intracellular Ca{sup 2+} levels.

  14. Cytosolic activity of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1A.

    Science.gov (United States)

    Livne, Sivan; Weiss, David

    2014-10-01

    Arabidopsis has three gibberellin receptors, GIBBERELLIN INSENSITIVE DWARF1 A (GID1A), GID1B and GID1C. All GID1s are localized to the nucleus but have also been detected in the cytoplasm. Since other major gibberellin signaling components have been localized to the nucleus, the current model suggests that gibberellin perception and signaling is nuclear. In this work we investigated whether GID1A can perceive gibberellin and initiate a signaling cascade in the cytosol. We have generated transgenic plants expressing GID1A fused to green florescence protein (GFP) and either a nuclear export signal (NES) or a nuclear localization signal (NLS), in the background of the gid1a gid1c (gid1ac) double mutant. Analyses by confocal microscopy confirmed the localization of GFP-GID1A-NES only in the cytosol and of GFP-GID1A-NLS only in the nucleus. Surprisingly, both recombinant proteins were able to complement the gid1ac mutation and to restore growth and responsiveness to gibberellin. However, transgenic seeds and seedlings expressing GFP-GID1A-NLS were more sensitive to gibberellin than those expressing GFP-GID1A-NES. The results of this study suggest that GID1A can bind gibberellin in the cytosol to initiate gibberellin signaling and responses. It is possible that activated cytosolic GID1A interacts with DELLA proteins before they enter the nucleus and induces a conformational transition that leads to DELLA's deactivation or degradation in the nucleus. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    OpenAIRE

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicit...

  16. Mediator-assisted Simultaneous probing of Cytosolic and Mitochondrial Redox activity in living cells

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Spegel, Christer; Kostesha, Natalie

    2009-01-01

    This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using...... either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pen-rose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric...

  17. Metformin Restores Parkin-Mediated Mitophagy, Suppressed by Cytosolic p53

    Directory of Open Access Journals (Sweden)

    Young Mi Song

    2016-01-01

    Full Text Available Metformin is known to alleviate hepatosteatosis by inducing 5’ adenosine monophosphate (AMP-kinase-independent, sirtuin 1 (SIRT1-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1 ad libitum feeding of a standard chow diet; (2 intraperitoneal injections of metformin 300 mg/kg; and (3 3 g/day caloric restriction (CR. HepG2 cells were treated with palmitate (PA plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins.

  18. Effect of Cyclosporin A and Angiotensin II on cytosolic calcium levels in primary human gingival fibroblasts

    Directory of Open Access Journals (Sweden)

    Ajitkumar Supraja

    2016-01-01

    Full Text Available Background: To evaluate the effect of Cyclosporin A (CsA and angiotensin II (Ang II on cytosolic calcium levels in cultured human gingival fibroblasts (HGFs. Materials and Methods: Healthy gingival samples from six volunteers were obtained, and primary HGFs were cultured. Cell viability and proliferation assay were performed to identify the ideal concentrations of CsA and Ang II. Cytosolic calcium levels in cultured gingival fibroblasts treated with CsA and Ang II were studied using colorimetric assay, confocal and fluorescence imaging. Statistical analyses were done using SPSS software and GraphPad Prism. Results: Higher levels of cytosolic levels were evident in cells treated with CsA and Ang II when compared to control group and was statistically significant (P < 0.05 in both colorimetric assay and confocal imaging. Fluorescent images of the cultured HGFs revealed the same. Conclusion: Thus calcium being a key player in major cellular functions, plays a major role in the pathogenesis of drug-induced gingival overgrowth.

  19. Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol

    Directory of Open Access Journals (Sweden)

    Harmen M. van Rossum

    2016-05-01

    Full Text Available In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1, nuclear-mitochondrial communication (RTG2, and encoding a carnitine acetyltransferase (YAT2. Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle.

  20. Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase.

    Science.gov (United States)

    Byrne, P C; Sanders, P G; Snell, K

    1992-08-15

    A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template. A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5' end and 653 bp at the 3' end. The full-length cDNA was cloned into an expression vector and transfected into COS-1 cells. Serine hydroxymethyltransferase activity was increased by 33% in the transfected cells and a new protein band of the appropriate size was seen by SDS/PAGE analysis of proteins extracted from transfected cells. The protein sequence for rabbit cytosolic serine hydroxymethyltransferase derived from the cDNA nucleotide sequence was compared with three other derived or known prokaryotic and eukaryotic sequences. An overall sequence similarity of 34% was noted between all four sequences, whereas the similarity between the rabbit cytosolic and mitochondrial isoforms was 62%.

  1. Cytosolic acidification as a signal mediating hyperosmotic stress responses in Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Klein Gérard

    2001-06-01

    Full Text Available Abstract Background Dictyostelium cells exhibit an unusual response to hyperosmolarity that is distinct from the response in other organisms investigated: instead of accumulating compatible osmolytes as it has been described for a wide range of organisms, Dictyostelium cells rearrange their cytoskeleton and thereby build up a rigid network which is believed to constitute the major osmoprotective mechanism in this organism. To gain more insight into the osmoregulation of this amoeba, we investigated physiological processes affected under hyperosmotic conditions in Dictyostelium. Results We determined pH changes in response to hyperosmotic stress using FACS or 31P-NMR. Hyperosmolarity was found to acidify the cytosol from pH 7.5 to 6.8 within 5 minutes, whereas the pH of the endo-lysosomal compartment remained constant. Fluid-phase endocytosis was identified as a possible target of cytosolic acidification, as the inhibition of endocytosis observed under hypertonic conditions can be fully attributed to cytosolic acidification. In addition, a deceleration of vesicle mobility and a decrease in the NTP pool was observed. Conclusion Together, these results indicate that hyperosmotic stress triggers pleiotropic effects, which are partially mediated by a pH signal and which all contribute to the downregulation of cellular activity. The comparison of our results with the effect of hyperosmolarity and intracellular acidification on receptor-mediated endocytosis in mammalian cells reveals striking similarities, suggesting the hypothesis of the same mechanism of inhibition by low internal pH.

  2. Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

    Science.gov (United States)

    Vita, Nicolas; Landolfi, Gianpiero; Baslé, Arnaud; Platsaki, Semeli; Lee, Jaeick; Waldron, Kevin J.; Dennison, Christopher

    2016-12-01

    Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis. These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1-2) × 1017 M-1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria.

  3. Tensegrity behaviour of cortical and cytosolic cytoskeletal components in twisted living adherent cells.

    Science.gov (United States)

    Laurent, Valérie M; Cañadas, Patrick; Fodil, Redouane; Planus, Emmanuelle; Asnacios, Atef; Wendling, Sylvie; Isabey, Daniel

    2002-01-01

    The present study is an attempt to relate the multicomponent response of the cytoskeleton (CSK), evaluated in twisted living adherent cells, to the heterogeneity of the cytoskeletal structure--evaluated both experimentally by means of 3D reconstructions, and theoretically considering the predictions given by two tensegrity models composed of (four and six) compressive elements and (respectively 12 and 24) tensile elements. Using magnetic twisting cytometry in which beads are attached to integrin receptors linked to the actin CSK of living adherent epithelial cells, we specifically measured the elastic CSK response at quasi equilibrium state and partitioned this response in terms of cortical and cytosolic contributions with a two-component model (i.e., a series of two Voigt bodies). These two CSK components were found to be prestressed and exhibited a stress-hardening response which both characterize tensegrity behaviour with however significant differences: compared to the cytosolic component, the cortical cytoskeleton appears to be a faster responding component, being a less prestressed and easily deformable structure. The discrepancies in elastic behaviour between the cortical and cytosolic CSK components may be understood on the basis of prestress tensegrity model predictions, given that the length and number of constitutive actin elements are taken into account.

  4. Structural Studies on Cytosolic Domain of Magnesium Transporter MgtE from Enterococcus faecalis

    Energy Technology Data Exchange (ETDEWEB)

    Ragumani, S.; Sauder, J; Burley, S; Swaminathan, S

    2009-01-01

    Magnesium (Mg{sup 2+}) is an essential element for growth and maintenance of living cells. It acts as a cofactor for many enzymes and is also essential for stability of the plasma membrane. There are two distinct classes of magnesium transporters identified in bacteria that convey Mg{sup 2+} from periplasm to cytoplasm [ATPase-dependent (MgtA and MgtB) and constitutively active (CorA and MgtE)]. Previously published work on Mg{sup 2+} transporters yielded structures of full length MgtE from Thermus thermophilus, determined at 3.5 {angstrom} resolution, and its cytoplasmic domain with and without bond Mg{sup 2+} determined at 2.3 and 3.9 {angstrom} resolution, respectively. Here, they report the crystal structure of the Mg{sup 2+} bound form of the cytosolic portion of MgtE (residues 6-262) from Enterococcus faecalis at 2.2 {angstrom} resolution. The present structure and magnesium bound cytosolic domain structure from T. thermophilus (PDB ID: 2YVY) are structurally similar. Three magnesium binding sites are common to both MgtE full length and the present structure. Their work revealed an additional Mg{sup 2+} binding site in the E. faecalis structure. In this report, they discuss the functional significance of Mg{sup 2+} binding sites in the cytosolic domains of MgtE transporters.

  5. Sulfation of melatonin: enzymatic characterization, differences of organs, species and genders, and bioactivity variation.

    Science.gov (United States)

    Tian, Xiangge; Huo, Xiaokui; Dong, Peipei; Wu, Baojian; Wang, Xiaobo; Wang, Chao; Liu, Kexin; Ma, Xiaochi

    2015-04-15

    Exogenous melatonin (Mel) is widely used in clinic for multiple therapeutic purposes. In metabolism pathways of Mel, 6-hydroxymelatonin-sulfate (S-O-Mel) and N-acetylserotonin sulfate (S-NAS) are the most abundant metabolites account for over 90% of total Mel metabolites in humans, indicating that sulfation plays an important role in reflecting the functions and clearance of Mel in vivo. In the present study, we characterized Mel sulfation using various human organ cytosols (liver, lung, kidney, small intestine and brain), liver cytosols from five different animal species, and cDNA-expressed human sulfotransferase (SULT) for the first time. Our results demonstrated that liver, lung, kidney and small intestine of humans had high catalytic efficiency for Mel sulfation, however, brain contained a very low reaction rate. Interestingly, organ cytosols prepared from females exhibited higher sulfation activity than those of males. SULT isoforms 1A1, 1A2, 1A3, 1B1 and 1E1 exhibited metabolic activities toward Mel. According to kinetic parameters (Km and Vmax), chemical inhibition, correlation analysis, molecular docking and sulfation assays with recombinant human SULTs isoforms, SULT1A1 was determined as the major enzyme responsible for Mel sulfation. Furthermore, considerable species differences in Mel sulfation were observed, and the total intrinsic clearance rate of Mel sulfation was as follows: monkey>rat>dog>human>pig>mouse. Additionally, the anti-inflammatory effects of Mel and its sulfated metabolites were evaluated by inhibiting nitric oxide (NO) production in RAW264.7 cells, and S-O-Mel as a bioactive form, exhibited potent bioactivity. Our investigation provided a global view of the enzyme-dependent sulfation of Mel that can guide biomedical research on Mel. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol.

    Science.gov (United States)

    van Rossum, Harmen M; Kozak, Barbara U; Niemeijer, Matthijs S; Dykstra, James C; Luttik, Marijke A H; Daran, Jean-Marc G; van Maris, Antonius J A; Pronk, Jack T

    2016-05-03

    In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle. This study demonstrates, for the first time, that Saccharomyces cerevisiae can be engineered to employ the carnitine shuttle for export of acetyl moieties from the mitochondria and, thereby, to act as the sole source of cytosolic acetyl-CoA. Further optimization of this ATP-independent mechanism for cytosolic acetyl-CoA provision can contribute to efficient

  7. Depletion of inhibitory factors against lipid peroxidation in cytosols of a radiation-sensitive mutant of L5178Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Nakazawa, T.; Yukawa, O.; Nagatsuka, S.; Matsudaira, H.; Sato, K. (National Inst. of Radiological Sciences, Chiba (Japan))

    1982-11-01

    The radiation-induced lipid peroxidation in soyabean lecithin liposomes was examined with and without various concentration of cytosols from several kinds of cells. After ..gamma..-irradiation the lipid peroxidation was determined as malondialdehyde (MDA) by the 2-thiobarbituric acid reaction according to Hunter et al. (1963). Without irradiation, there was no change in the MDA level of liposomes, after the addition of low concentrations of cytosols, but a gradual increase of the MDA level was caused in the presence of higher cytosol concentrations in liver and in M10 cells, though not in L5178Y cells. There was no recognisable MDA in cytosol even at the higher concentrations, either immediately or more than one hour after irradiation.

  8. Light‐dependent regulation of ascorbate in tomato by a monodehydroascorbate reductase localized in peroxisomes and the cytosol

    National Research Council Canada - National Science Library

    Gest, Noé; Garchery, Cécile; Gautier, Hélène; Jiménez, Ana; Stevens, Rebecca

    2013-01-01

    .... In plants, MDHAR belongs to a multigene family. Here, we report the presence of an MDHAR isoform in both the cytosol and peroxisomes and show that this enzyme negatively regulates ascorbate levels in Solanum lycopersicum (tomato...

  9. Recombinant BCG Expressing ESX-1 of Mycobacterium marinum Combines Low Virulence with Cytosolic Immune Signaling and Improved TB Protection

    NARCIS (Netherlands)

    Gröschel, Matthias I.; Sayes, Fadel; Shin, Sung Jae; Frigui, Wafa; Pawlik, Alexandre; Orgeur, Mickael; Canetti, Robin; Honore, Nadine; Simeone, Roxane; van der Werf, Tjip S.; Bitter, Wilbert; Cho, Sang-Nae; Majlessi, Laleh; Brosch, Roland

    2017-01-01

    Recent insights into the mechanisms by which Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is recognized by cytosolic nucleotide sensors have opened new avenues for rational vaccine design. The only licensed anti-tuberculosis vaccine, Mycobacteriumbovis BCG, provides limited

  10. The role and interactions of cytosolic alkalization and hydrogen peroxide in ultraviolet B-induced stomatal closure in Arabidopsis.

    Science.gov (United States)

    Zhu, Yan; Ge, Xiao-Min; Wu, Mi-Mi; Li, Xuan; He, Jun-Min

    2014-02-01

    Cytosolic alkalization has been shown to function as a key player in multiple stimuli-induced stomatal closure, but its role and relationship with hydrogen peroxide (H2O2) in ultraviolet B (UV-B)-induced stomatal closure remains unknown. In this paper, by stomatal bioassay and laser-scanning confocal microscopy, we observed that 0.5 W m(-2) UV-B induced cytosolic alkalization and H2O2 production in guard cells while inducing stomatal closure in Arabidopsis (Arabidopsis thaliana). Butyrate (a weak acid) reduced the cytosolic pH/H2O2 production and prevented stomatal closure by UV-B. Methylamine (a weak base) induced H2O2 production and stomatal closure while enhancing the cytosolic alkalization in guard cells under light alone. The rise in cytosolic pH of wild-type guard cells on exposure to UV-B was evident at 15 min and substantial at 45 min while H2O2 production started to largely increase after 60 min. The failure of UV-B-induced H2O2 production in AtrbohD/F guard cells did not affect the changes of guard cell pH during the first 60 min of UV-B radiation, but largely suppressed cytosolic alkalization after 60 min of UV-B radiation. These results indicate that cytosolic alkalization mediates UV-B-induced stomatal closure via activating H2O2 production and that H2O2 production can feedback-enhance cytosolic alkalization in Arabidopsis guard cells. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  11. Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio [Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias s/n, 15782 Santiago de Compostela (Spain); Bermejo-Barrera, Pilar [Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias s/n, 15782 Santiago de Compostela (Spain)], E-mail: pbermejo@usc.es; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad [Department of Analytical Chemistry, Faculty of Sciences, University of A Coruna, Campus da Zapateira s/n, 15071 A Coruna (Spain); Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario [Department of Analytical Chemistry, Faculty of Sciences, University of A Coruna, Campus da Zapateira s/n, 15071 A Coruna (Spain); University Institute of Environment, University of A Coruna, Pazo de Longora, Lians, 15179 Oleiros (Spain)

    2007-11-05

    The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices.

  12. A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival.

    Science.gov (United States)

    Chen, Grischa Y; McDougal, Courtney E; D'Antonio, Marc A; Portman, Jonathan L; Sauer, John-Demian

    2017-03-21

    Through unknown mechanisms, the host cytosol restricts bacterial colonization; therefore, only professional cytosolic pathogens are adapted to colonize this host environment. Listeria monocytogenes is a Gram-positive intracellular pathogen that is highly adapted to colonize the cytosol of both phagocytic and nonphagocytic cells. To identify L. monocytogenes determinants of cytosolic survival, we designed and executed a novel screen to isolate L. monocytogenes mutants with cytosolic survival defects. Multiple mutants identified in the screen were defective for synthesis of menaquinone (MK), an essential molecule in the electron transport chain. Analysis of an extensive set of MK biosynthesis and respiratory chain mutants revealed that cellular respiration was not required for cytosolic survival of L. monocytogenes but that, instead, synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an MK biosynthesis intermediate, was essential. Recent discoveries showed that modulation of the central metabolism of both host and pathogen can influence the outcome of host-pathogen interactions. Our results identify a potentially novel function of the MK biosynthetic intermediate DHNA and specifically highlight how L. monocytogenes metabolic adaptations promote cytosolic survival and evasion of host immunity.IMPORTANCE Cytosolic bacterial pathogens, such as Listeria monocytogenes and Francisella tularensis, are exquisitely evolved to colonize the host cytosol in a variety of cell types. Establishing an intracellular niche shields these pathogens from effectors of humoral immunity, grants access to host nutrients, and is essential for pathogenesis. Through yet-to-be-defined mechanisms, the host cytosol restricts replication of non-cytosol-adapted bacteria, likely through a combination of cell autonomous defenses (CADs) and nutritional immunity. Utilizing a novel genetic screen, we identified determinants of L. monocytogenes cytosolic survival and virulence and identified a role for

  13. Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

    Directory of Open Access Journals (Sweden)

    Corinna Hiller

    2014-04-01

    Full Text Available African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective

  14. A cytosolic juxtamembrane interface modulates plexin A3 oligomerization and signal transduction.

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    Rachael Barton

    Full Text Available Plexins (plxns are transmembrane (TM receptors involved in the guidance of vascular, lymphatic vessel, and neuron growth as well as cancer metastasis. Plxn signaling results in cytosolic GTPase-activating protein activity, and previous research implicates dimerization as important for activation of plxn signaling. Purified, soluble plxn extracellular and cytosolic domains exhibit only weak homomeric interactions, suggesting a role for the plxn TM and juxtamembrane regions in homooligomerization. In this study, we consider a heptad repeat in the Danio rerio PlxnA3 cytosolic juxtamembrane domain (JM for its ability to influence PlxnA3 homooligomerization in TM-domain containing constructs. Site-directed mutagenesis in conjunction with the AraTM assay and bioluminescent energy transfer (BRET² suggest an interface involving a JM heptad repeat, in particular residue M1281, regulates PlxnA3 homomeric interactions when examined in constructs containing an ectodomain, TM and JM domain. In the presence of a neuropilin-2a co-receptor and semaphorin 3F ligand, disruption to PlxnA3 homodimerization caused by an M1281F mutation is eliminated, suggesting destabilization of the PlxnA3 homodimer in the JM is not sufficient to disrupt co-receptor complex formation. In contrast, enhanced homodimerization of PlxnA3 caused by mutation M1281L remains even in the presence of ligand semaphorin 3F and co-receptor neuropilin-2a. Consistent with this pattern of PlxnA3 dimerization in the presence of ligand and co-receptor, destabilizing mutations to PlxnA3 homodimerization (M1281F are able to rescue motor patterning defects in sidetracked zebrafish embryos, whereas mutations that enhance PlxnA3 homodimerization (M1281L are not. Collectively, our results indicate the JM heptad repeat, in particular residue M1281, forms a switchable interface that modulates both PlxnA3 homomeric interactions and signal transduction.

  15. The role of a cytosolic superoxide dismutase in barley-pathogen interactions

    KAUST Repository

    Lightfoot, Damien

    2016-03-19

    Reactive oxygen species (ROS), including superoxide (O2-HO2) and hydrogen peroxide (H2O2), are differentially produced during resistance responses to biotrophic pathogens and during susceptible responses to necrotrophic and hemi-biotrophic pathogens. Superoxide dismutase (SOD) is responsible for the catalysis of the dismutation of O2-HO2 to H2O2, regulating the redox status of plant cells. Increased SOD activity has been correlated previously with resistance in barley to the hemi-biotrophic pathogen Pyrenophora teres f. teres (Ptt, the causal agent of the net form of net blotch disease), but the role of individual isoforms of SOD has not been studied. A cytosolic CuZnSOD, HvCSD1, was isolated from barley and characterized as being expressed in tissue from different developmental stages. HvCSD1 was up-regulated during the interaction with Ptt and to a greater extent during the resistance response. Net blotch disease symptoms and fungal growth were not as pronounced in transgenic HvCSD1 knockdown lines in a susceptible background (cv. Golden Promise), when compared with wild-type plants, suggesting that cytosolic O2-HO2 contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi-biotrophic rice blast pathogen Magnaporthe oryzae or the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei, but HvCSD1 also played a role in the regulation of lesion development by methyl viologen. Together, these results suggest that HvCSD1 could be important in the maintenance of the cytosolic redox status and in the differential regulation of responses to pathogens with different lifestyles.

  16. Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing.

    Science.gov (United States)

    Mout, Rubul; Ray, Moumita; Yesilbag Tonga, Gulen; Lee, Yi-Wei; Tay, Tristan; Sasaki, Kanae; Rotello, Vincent M

    2017-03-28

    Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.

  17. Localization and function of cytosolic phospholipase A2α at the Golgi

    OpenAIRE

    Leslie, Christina C.; Gangelhoff, Todd A.; Gelb, Michael H.

    2010-01-01

    Cytosolic phospholipase A2α (cPLA2α, Group IVA phospholipase A2) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA2α translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme’s calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA2α translocation to the Golgi. However, other features of cPLA2α regulation are incompletely un...

  18. Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

    DEFF Research Database (Denmark)

    Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

    2009-01-01

    factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

  19. Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans

    OpenAIRE

    Černila, Boštjan; Črešnar, Bronislava; Breskvar, Katja

    2003-01-01

    Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any addit...

  20. Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase.

    OpenAIRE

    Byrne, P C; Sanders, P. G.; Snell, K

    1992-01-01

    A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template. A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5' end and 653 bp at the 3' end. The...

  1. Effective Selection of a Well-Differentiated Type of Human Uterine Endometrial Carcinoma Cells by Transfection of the Sulfotransferase Gene and Possible Association of Sulfoglycolipids With Well-Differentiated Phenotypes.

    Science.gov (United States)

    Tanaka, Kyoko; Ishiwata, Isamu; Kubushiro, Kaneyuki; Mikami, Mikio; Aoki, Daisuke; Kiguchi, Kazushige; Iwamori, Masao

    2017-02-01

    Sulfatide has been shown to be characteristically increased on the apical surface of the normal endometrium at the secretory phase, and to be related with the formation of the glandular structure and the secretion of mucin from glands for the implantation of a fertilized egg. Additionally, sulfatides are expressed in the well-differentiated type, but not in the poorly differentiated type, of endometrial carcinomas. This suggests that sulfatides are a molecular marker of differentiated phenotypes. To further elucidate the biological significance of sulfoglycolipids, we transfected the sulfotransferase gene into endometrial carcinoma-derived cells without sulfoglycolipids and compared their glycolipid compositions and phenotypes with those of the original cells. The glycolipid sulfotransferase gene was transfected into endometrial carcinoma-derived SNG-II cells, the resultant transfected cells being found to frequently form a domelike structure, and some of them were selected as SNG-II-GST cells. We compared the glycolipid compositions and phenotypes of SNG-II and SNG-II-GST cells. Although the original SNG-II cells grew in a paving stone pattern, SNG-II-GST cells formed a domelike structure. SNG-II-GST cells exhibited high GST activity and contained sulfoglycolipids, IISO3-LacCer and IISO3-Gg3Cer, which were not found in SNG-II cells. The amounts of sulfoglycolipids in SNG-II-GST cells were 1.5 times higher than those of gangliosides, and the proportions of LacCer and GM3 in SNG-II-GST cells were greatly different from those in SNG-II cells. SNG-II and SNG-II GST cells exhibited poorly differentiated and well-differentiated phenotypes on histochemical examination of cancerous nodules in nude mice. However, by means of an oxygen electrode, SNG-II-GST cells were found to be more resistant to anticancer drugs than SNG-II cells. Enhanced expression of sulfoglycolipids in poorly differentiated cells is a feasible means of selecting well-differentiated ones, and

  2. Targeting cytosolic proliferating cell nuclear antigen (PCNA in neutrophil-dominated inflammation

    Directory of Open Access Journals (Sweden)

    Alessia eDe Chiara

    2012-10-01

    Full Text Available New therapeutic approaches that can accelerate neutrophil apoptosis under inflammatory conditions to enhance the resolution of inflammation are now under study. Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA, a nuclear factor involved in DNA replication and repair of proliferating cells is a key regulator of neutrophil survival. In this review, we will try to put into perspective the physiologic relevance of PCNA in neutrophils. We will discuss key issues such as molecular structure, post-translational modifications, based on our knowledge of nuclear PCNA, assuming that similar principles governing its function are conserved between nuclear and cytosolic PCNA. The example of cystic fibrosis that features one of the most intense neutrophil-dominated pulmonary inflammation will be discussed. We believe that through an intimate comprehension of the cytosolic PCNA scaffold based on nuclear PCNA knowledge, novel pathways regulating neutrophil survival can be unraveled and innovative agents can be developed to dampen inflammation where it proves detrimental.

  3. Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes.

    Directory of Open Access Journals (Sweden)

    Marzia Massignani

    Full Text Available BACKGROUND: Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. PRINCIPAL FINDINGS: We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. SIGNIFICANCE: We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Se Jeong [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Gu, Dong Ryun [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Jin, Su Hyun [Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Park, Keun Ha [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Lee, Seoung Hoon, E-mail: leesh2@wku.ac.kr [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Wonkwang Institute of Biomaterials and Implant, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of)

    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.

  5. Characterization of protein quality control components via dual reporter-containing misfolded cytosolic model substrates.

    Science.gov (United States)

    Amm, Ingo; Kawan, Mona; Wolf, Dieter H

    2016-12-15

    Protein misfolding and protein aggregation are causes of severe diseases as neurodegenerative disorders, diabetes and cancer. Therefore, the cell has to constantly monitor the folding status of its proteome. Chaperones and components of the ubiquitin-proteasome system are key players in the cellular protein quality control process. In order to characterize components of the protein quality control system in a well-established model eukaryote - the yeast Saccharomyces cerevisiae - we established new cytosolic model substrates based on firefly luciferase and β-isopropylmalate dehydrogenase (Leu2). The use of these two different enzymes arranged in tandem as reporters enabled us to analyse the folding status and the degradation propensity of these new model substrates in yeast cells mutated in components of the cellular protein quality control system. The Hsp70 chaperone system known to be essential in the cellular protein quality control was chosen as a model for showing the high value of the luciferase-based model substrates in the characterization of components of the cytosolic protein quality control system in yeast. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Sherley, J.L.; Kelly, T.J.

    1988-01-05

    The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity.

  7. Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis.

    Science.gov (United States)

    Ogawa, Takahisa; Kimura, Ayako; Sakuyama, Harumi; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

    2015-01-01

    Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The Km and Vmax values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min(-1) mg protein(-1), respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.

  8. Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris

    Directory of Open Access Journals (Sweden)

    Zhengyi Liu

    2014-01-01

    Full Text Available Heat shock protein 90 (HSP90, a highly conserved molecular chaperone, plays essential roles in folding, keeping structural integrity, and regulating the subset of cytosolic proteins. We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90 by combining homology cloning with rapid amplification of cDNA ends (RACE. Sequence analysis indicated that CvHSP90 is a cytosolic member of the HSP90 family. Quantitative RT-PCR was applied to determine the expression level of messenger RNA (mRNA in CvHSP90 under different stress conditions. C. vulgaris was kept in different temperatures (5–45°C for 1 h. The mRNA expression level of CvHSP90 increased with temperature from 5 to 10°C, went further from 35 to 40°C, and reached the maximum at 40°C. On the other hand, for C. vulgaris kept at 35°C for different durations, the mRNA expression level of CvHSP90 increased gradually and reached the peak at 7 h and then declined progressively. In addition, the expression level of CvHSP90 at 40 or 45 in salinity (‰ was almost fourfold of that at 25 in salinity (‰ for 2 h. Therefore, CvHSP90 may be a potential biomarker to monitor environment changes.

  9. A cytosolic activator of DNA replication is tyrosine phosphorylated in its active form.

    Science.gov (United States)

    Fresa, K L; Autieri, M V; Coffman, F D; Georgoff, I; Cohen, S

    1993-04-01

    Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a > 90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from non-proliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself.

  10. Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans.

    Science.gov (United States)

    Cernila, Bostjan; Cresnar, Bronislava; Breskvar, Katja

    2003-01-01

    Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any additional highly homologous copies of the cytosolic hsp70 genes in the R nigricans genome. Sequence analyses showed that all 3 genes contain introns within the open reading frame. The dynamics of the R nigricans molecular response to progesterone, 21-OH progesterone, and testosterone, as well as to heat shock, copper ions, hydrogen peroxide, and ethanol was studied by temporal analysis of Rnhsp70-1 and Rnhsp70-2 mRNA accumulation. Northern blot experiments revealed that the Rnhsp70-2 transcript level is not affected by testosterone, whereas mRNA levels of both genes are rapidly increased with all the other stressors studied. Moreover, the decrease of transcript levels is notably delayed in ethanol stress, and a difference is observed between the profiles of Rnhsp70-1 and Rnhsp70-2 transcripts during heat stress.

  11. Cytosolic RNA:DNA hybrids activate the cGAS-STING axis.

    Science.gov (United States)

    Mankan, Arun K; Schmidt, Tobias; Chauhan, Dhruv; Goldeck, Marion; Höning, Klara; Gaidt, Moritz; Kubarenko, Andrew V; Andreeva, Liudmila; Hopfner, Karl-Peter; Hornung, Veit

    2014-12-17

    Intracellular recognition of non-self and also self-nucleic acids can result in the initiation of potent pro-inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2'-5'), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP-1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS-STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA. © 2014 The Authors.

  12. Cytosolic localization of acetohydroxyacid synthase Ilv2 and its impact on diacetyl formation during beer fermentation.

    Science.gov (United States)

    Dasari, Suvarna; Kölling, Ralf

    2011-02-01

    Diacetyl (2,3-butanedione) imparts an unpleasant "butterscotch-like" flavor to alcoholic beverages such as beer, and therefore its concentration needs to be reduced below the sensory threshold before packaging. We examined the mechanisms that lead to highly elevated diacetyl formation in petite mutants of Saccharomyces cerevisiae during beer fermentations. We present evidence that elevated diacetyl formation is tightly connected to the mitochondrial import of acetohydroxyacid synthase (Ilv2), the key enzyme in the production of diacetyl. Our data suggest that accumulation of the matrix-targeted Ilv2 preprotein in the cytosol is responsible for the observed high diacetyl levels. We could show that the Ilv2 preprotein accumulates in the cytosol of petite yeasts. Furthermore, expression of an Ilv2 variant that lacks the N-terminal mitochondrial targeting sequence and thus cannot be imported into mitochondria led to highly elevated diacetyl levels comparable to a petite strain. We further show that expression of a mutant allele of the γ-subunit of the F(1)-ATPase (ATP3-5) could be an attractive way to reduce diacetyl formation by petite strains.

  13. Profiling of cytosolic and peroxisomal acetyl-CoA metabolism in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yun Chen

    Full Text Available As a key intracellular metabolite, acetyl-coenzyme A (acetyl-CoA plays a major role in various metabolic pathways that link anabolism and catabolism. In the yeast Saccharomyces cerevisiae, acetyl-CoA involving metabolism is compartmentalized, and may vary with the nutrient supply of a cell. Membranes separating intracellular compartments are impermeable to acetyl-CoA and no direct transport between the compartments occurs. Thus, without carnitine supply the glyoxylate shunt is the sole possible route for transferring acetyl-CoA from the cytosol or the peroxisomes into the mitochondria. Here, we investigate the physiological profiling of different deletion mutants of ACS1, ACS2, CIT2 and MLS1 individually or in combination under alternative carbon sources, and study how various mutations alter carbon distribution. Based on our results a detailed model of carbon distribution about cytosolic and peroxisomal acetyl-CoA metabolism in yeast is suggested. This will be useful to further develop yeast as a cell factory for the biosynthesis of acetyl-CoA-derived products.

  14. Differential modulation of cytosolic lipases activities in liver and adipose tissue by high-carbohydrate diets.

    Science.gov (United States)

    Rodrigues, Angélica Heringer; Moreira, Carolina Campos Lima; Mario, Érica Guilhen; de Souza Cordeiro, Letícia Maria; Avelar, Gleide Fernandes; Botion, Leida Maria; Chaves, Valéria Ernestânia

    2016-08-01

    Several studies have demonstrated that a high-fructose (FRUC) diet induces metabolic and haemodynamic abnormalities, known as the metabolic syndrome, which are characterised by obesity, glucose intolerance, insulin resistance, dyslipidaemia and hypertension. In this study, the effect of a FRUC diet (60 % fructose) for 8 weeks on the metabolism of lipids in liver and epididymal adipose tissue from Wistar rats was compared with the AIN-93M diet and the effects of the AIN-93M diet were compared with a chow diet. The FRUC diet induced marked increases in both hepatocyte lipid droplet volume and postprandial serum levels of triacylglycerol (TAG), but reduced the postprandial serum levels of insulin. The AIN-93M diet induced marked increases in the hepatocyte lipid droplet volume and the serum levels of insulin, without affecting the serum levels of TAG. We found that isocaloric substitution of cornstarch, dextrinised cornstarch and sucrose (AIN-93M diet) for fructose did not affect the hepatic VLDL-TAG secretion and adipose tissue glucose uptake, lipolysis and cytosolic lipases activities in rats. However, the high-fructose diet induced a severe steatosis in liver accompanied by a decrease in cytosolic lipases activities. In adipose tissue, the FRUC diet induced a decrease in the lipoprotein lipase activity, and an increase in lipogenesis. FRUC and AIN-93M diets induced changes in lipid homeostasis in liver and adipose tissue by distinct biochemical mechanisms.

  15. MLL5 maintains spindle bipolarity by preventing aberrant cytosolic aggregation of PLK1.

    Science.gov (United States)

    Zhao, Wei; Liu, Jie; Zhang, Xiaoming; Deng, Lih-Wen

    2016-03-28

    Faithful chromosome segregation with bipolar spindle formation is critical for the maintenance of genomic stability. Perturbation of this process often leads to severe mitotic failure, contributing to tumorigenesis. MLL5 has been demonstrated to play vital roles in cell cycle progression and the maintenance of genomic stability. Here, we identify a novel interaction between MLL5 and PLK1 in the cytosol that is crucial for sustaining spindle bipolarity during mitosis. Knockdown of MLL5 caused aberrant PLK1 aggregation that led to acentrosomal microtubule-organizing center (aMTOC) formation and subsequent spindle multipolarity. Further molecular studies revealed that the polo-box domain (PBD) of PLK1 interacted with a binding motif on MLL5 (Thr887-Ser888-Thr889), and this interaction was essential for spindle bipolarity. Overexpression of wild-type MLL5 was able to rescue PLK1 mislocalization and aMTOC formation in MLL5-KD cells, whereas MLL5 mutants incapable of interacting with the PBD failed to do so. We thus propose that MLL5 preserves spindle bipolarity through maintaining cytosolic PLK1 in a nonaggregated form. © 2016 Zhao et al.

  16. Expression and characterization of a cytosolic glucose 6 phosphate dehydrogenase isoform from barley (Hordeum vulgare) roots.

    Science.gov (United States)

    Castiglia, Daniela; Cardi, Manuela; Landi, Simone; Cafasso, Donata; Esposito, Sergio

    2015-08-01

    In plant cells, glucose 6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) regulates the oxidative pentose phosphate pathway (OPPP), a metabolic route involved in the production of NADPH for various biosynthetic processes and stress response. In this study, we report the overexpression of a cytosolic G6PDH isoform from barley (Hordeum vulgare) roots in bacteria, and the biochemical characterization of the purified recombinant enzyme (HvCy-G6PDH). A full-length cDNA coding for a cytosolic isoform of G6PDH was isolated, and the sequence was cloned into pET3d vector; the protein was overexpressed in Escherichia coli BL21 (DE3) and purified by anion exchange and affinity chromatography. The kinetic properties were calculated: the recombinant HvCy-G6PDH showed KMs and KINADPH comparable to those observed for the enzyme purified from barley roots; moreover, the analysis of NADPH inhibition suggested a competitive mechanism. Therefore, this enzyme could be utilised for the structural and regulatory characterization of this isoform in higher plants. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Cytosolic malate dehydrogenase activity helps support glycolysis in actively proliferating cells and cancer.

    Science.gov (United States)

    Hanse, E A; Ruan, C; Kachman, M; Wang, D; Lowman, X H; Kelekar, A

    2017-07-06

    Increased glucose consumption is a hallmark of cancer cells. The increased consumption and subsequent metabolism of glucose during proliferation creates the need for a constant supply of NAD, a co-factor in glycolysis. Regeneration of the NAD required to support enhanced glycolysis has been attributed to the terminal glycolytic enzyme, lactate dehydrogenase (LDH). However, loss of glucose carbons to biosynthetic pathways early in glycolysis reduces the carbon supply to LDH. Thus, alternative routes for NAD regeneration must exist to support the increased glycolytic rate while allowing for the diversion of glucose to generate biomass and support proliferation. Here we demonstrate, using a variety of cancer cell lines as well as activated primary T cells, that cytosolic malate dehydrogenase 1 (MDH1) is an alternative to LDH as a supplier of NAD. Moreover, our results indicate that MDH1 generates malate with carbons derived from glutamine, thus enabling utilization of glucose carbons for glycolysis and for biomass. Amplification of MDH1 occurs at an impressive frequency in human tumors and correlates with poor prognosis. Together, our findings suggest that proliferating cells rely on both MDH1 and LDH to replenish cytosolic NAD, and that therapies designed at targeting glycolysis must consider both dehydrogenases.

  18. Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans

    Science.gov (United States)

    Černila, Boštjan; Črešnar, Bronislava; Breskvar, Katja

    2003-01-01

    Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any additional highly homologous copies of the cytosolic hsp70 genes in the R nigricans genome. Sequence analyses showed that all 3 genes contain introns within the open reading frame. The dynamics of the R nigricans molecular response to progesterone, 21-OH progesterone, and testosterone, as well as to heat shock, copper ions, hydrogen peroxide, and ethanol was studied by temporal analysis of Rnhsp70-1 and Rnhsp70-2 mRNA accumulation. Northern blot experiments revealed that the Rnhsp70-2 transcript level is not affected by testosterone, whereas mRNA levels of both genes are rapidly increased with all the other stressors studied. Moreover, the decrease of transcript levels is notably delayed in ethanol stress, and a difference is observed between the profiles of Rnhsp70-1 and Rnhsp70-2 transcripts during heat stress. PMID:15115284

  19. Solution NMR of MPS-1 reveals a random coil cytosolic domain structure.

    Directory of Open Access Journals (Sweden)

    Pan Li

    Full Text Available Caenorhabditis elegans MPS1 is a single transmembrane helical auxiliary subunit that co-localizes with the voltage-gated potassium channel KVS1 in the nematode nervous system. MPS-1 shares high homology with KCNE (potassium voltage-gated channel subfamily E member auxiliary subunits, and its cytosolic domain was reported to have a serine/threonine kinase activity that modulates KVS1 channel function via phosphorylation. In this study, NMR spectroscopy indicated that the full length and truncated MPS-1 cytosolic domain (134-256 in the presence or absence of n-dodecylphosphocholine detergent micelles adopted a highly flexible random coil secondary structure. In contrast, protein kinases usually adopt a stable folded conformation in order to implement substrate recognition and phosphoryl transfer. The highly flexible random coil secondary structure suggests that MPS-1 in the free state is unstructured but may require a substrate or binding partner to adopt stable structure required for serine/threonine kinase activity.

  20. Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.

    Science.gov (United States)

    Kozak, Barbara U; van Rossum, Harmen M; Luttik, Marijke A H; Akeroyd, Michiel; Benjamin, Kirsten R; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

    2014-10-21

    The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy

  1. Reverse genetic characterization of cytosolic acetyl-CoA generation by ATP-citrate lyase in Arabidopsis.

    Science.gov (United States)

    Fatland, Beth L; Nikolau, Basil J; Wurtele, Eve Syrkin

    2005-01-01

    Acetyl-CoA provides organisms with the chemical flexibility to biosynthesize a plethora of natural products that constitute much of the structural and functional diversity in nature. Recent studies have characterized a novel ATP-citrate lyase (ACL) in the cytosol of Arabidopsis thaliana. In this study, we report the use of antisense RNA technology to generate a series of Arabidopsis lines with a range of ACL activity. Plants with even moderately reduced ACL activity have a complex, bonsai phenotype, with miniaturized organs, smaller cells, aberrant plastid morphology, reduced cuticular wax deposition, and hyperaccumulation of starch, anthocyanin, and stress-related mRNAs in vegetative tissue. The degree of this phenotype correlates with the level of reduction in ACL activity. These data indicate that ACL is required for normal growth and development and that no other source of acetyl-CoA can compensate for ACL-derived acetyl-CoA. Exogenous malonate, which feeds into the carboxylation pathway of acetyl-CoA metabolism, chemically complements the morphological and chemical alterations associated with reduced ACL expression, indicating that the observed metabolic alterations are related to the carboxylation pathway of cytosolic acetyl-CoA metabolism. The observations that limiting the expression of the cytosolic enzyme ACL reduces the accumulation of cytosolic acetyl-CoA-derived metabolites and that these deficiencies can be alleviated by exogenous malonate indicate that ACL is a nonredundant source of cytosolic acetyl-CoA.

  2. Compartment-specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol.

    Science.gov (United States)

    Matuszczyk, Jens-Christoph; Teleki, Attila; Pfizenmaier, Jennifer; Takors, Ralf

    2015-10-01

    Mammalian cells show a compartmented metabolism. Getting access to subcellular metabolite pools is of high interest to understand the cells' metabolomic state. Therefore a protocol is developed and applied for monitoring compartment-specific metabolite and nucleotide pool sizes in Chinese hamster ovary (CHO) cells. The approach consists of a subtracting filtering method separating cytosolic components from physically intact mitochondrial compartments. The internal standards glucose-6-phosphate and cis-aconitate were chosen to quantify cytosolic secession and mitochondrial membrane integrity. Extracts of related fractions were studied by liquid chromatography-isotope dilution mass spectrometry for the absolute quantification of a subset of glycolytic and tricarboxylic acid cycle intermediates together with the adenylate nucleotides ATP, ADP and AMP. The application of the protocol revealed highly dynamic changes in the related pool sizes as a function of distinct cultivation periods of IgG1 producing CHO cells. Mitochondrial and cytosolic pool dynamics were in agreement with anticipated metabolite pools of independent studies. The analysis of adenosine phosphate levels unraveled significantly higher ATP levels in the cytosol leading to the hypothesis that mitochondria predominantly serve for fueling ATP into the cytosol where it is tightly controlled at physiological adenylate energy charges about 0.9. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Glutathione redox potential in the mitochondrial intermembrane space is linked to the cytosol and impacts the Mia40 redox state

    Science.gov (United States)

    Kojer, Kerstin; Bien, Melanie; Gangel, Heike; Morgan, Bruce; Dick, Tobias P; Riemer, Jan

    2012-01-01

    Glutathione is an important mediator and regulator of cellular redox processes. Detailed knowledge of local glutathione redox potential (EGSH) dynamics is critical to understand the network of redox processes and their influence on cellular function. Using dynamic oxidant recovery assays together with EGSH-specific fluorescent reporters, we investigate the glutathione pools of the cytosol, mitochondrial matrix and intermembrane space (IMS). We demonstrate that the glutathione pools of IMS and cytosol are dynamically interconnected via porins. In contrast, no appreciable communication was observed between the glutathione pools of the IMS and matrix. By modulating redox pathways in the cytosol and IMS, we find that the cytosolic glutathione reductase system is the major determinant of EGSH in the IMS, thus explaining a steady-state EGSH in the IMS which is similar to the cytosol. Moreover, we show that the local EGSH contributes to the partially reduced redox state of the IMS oxidoreductase Mia40 in vivo. Taken together, we provide a comprehensive mechanistic picture of the IMS redox milieu and define the redox influences on Mia40 in living cells. PMID:22705944

  4. Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2.

    Science.gov (United States)

    Luyten, Tomas; Welkenhuyzen, Kirsten; Roest, Gemma; Kania, Elzbieta; Wang, Liwei; Bittremieux, Mart; Yule, David I; Parys, Jan B; Bultynck, Geert

    2017-06-01

    Previous work revealed that intracellular Ca2+ signals and the inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP3Rs and Ca2+ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca2+-chelating agent. To elucidate the IP3R's role in this process, we employed the recently established HEK 3KO cells lacking all three IP3R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP3R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca2+ or by knocking out IP3Rs. Finally, we investigated whether resveratrol by itself induced Ca2+ release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca2+ ATPase (SERCA) activity nor the IP3-induced Ca2+ release nor the basal Ca2+ leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP3-induced Ca2+ release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca2+ signals by itself, it acutely decreased the ER Ca2+-store content irrespective of the presence or absence of IP3Rs, leading to a dampened agonist-induced Ca2+ signaling. In conclusion, these results reveal that IP3Rs and cytosolic Ca2+ signaling are fundamentally important for driving autophagic flux, not only in response to m

  5. Changes in cytosolic pH and calcium of guard cells precede stomatal movements.

    Science.gov (United States)

    Irving, H R; Gehring, C A; Parish, R W

    1992-03-01

    Stomatal opening is induced by indoleacetic acid (IAA), cytokinins, and fusicoccin (FC), whereas stomatal closure is induced by abscisic acid (ABA). To test the effect of these growth regulators on guard cell cytosolic Ca2+ ([Ca2+]cyt) and pH (pHcyt), epidermal strips were taken from the lower side of leaves of the orchid Paphiopedilum tonsum and were loaded with acetomethoxy-esterified forms of the Ca2+ indicator fluo-3 or the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)carboxyfluorescein. Basal [Ca2+]cyt ranged from 0.05 to 0.3 M and was 0.22 +/- 0.015 (n = 21). Increases in both [Ca2+]cyt and pHcyt were observed in guard cells after application of 10-100 M ABA to open stomata, and these preceded stomatal closure. The increase in [Ca2+]cyt ranged from 1.5- to 3-fold and was seen in 7 of 10 experiments. Guard cell alkalinization began within 2 min of ABA treatment and continued for the next 8 min. The increase ranged from 0.04 to 0.3 pH unit and was seen in 13 of 14 experiments. Guard cell [Ca2+]cyt increased, whereas pHcyt decreased after treatment of closed stomata with IAA, kinetin, or FC. In response to 50-100 M IAA, [Ca2+]cyt increased 1.5- to 2-fold in all cases, and pHcyt decreased 0.2-0.4 unit within 5 min in 7 experiments. Within 12 min, 10-100 M kinetin caused [Ca2+]cyt to increase in 28 of 34 experiments (1.3- to 2.5-fold) and pHcyt fell 0.1-0.4 unit in 15 of 17 treatments. The response to 10-50 M FC was similar in both time and magnitude. These results show that stomatal opening is accompanied by an increase in [Ca2+]cyt and cytosolic acidification in the guard cells, whereas stomatal closure is preceded by an increase in [Ca2+]cyt and cytosolic alkalinization in the guard cells. The order of these events is still uncertain, but changes in pHcyt are correlated with stomatal movement, and these changes may be an important factor in the regulation of guard cell movement.

  6. Skeletal muscle contractions induce acute changes in cytosolic superoxide, but slower responses in mitochondrial superoxide and cellular hydrogen peroxide.

    Directory of Open Access Journals (Sweden)

    Timothy Pearson

    Full Text Available Skeletal muscle generation of reactive oxygen species (ROS is increased following contractile activity and these species interact with multiple signaling pathways to mediate adaptations to contractions. The sources and time course of the increase in ROS during contractions remain undefined. Confocal microscopy with specific fluorescent probes was used to compare the activities of superoxide in mitochondria and cytosol and the hydrogen peroxide content of the cytosol in isolated single mature skeletal muscle (flexor digitorum brevis fibers prior to, during, and after electrically stimulated contractions. Superoxide in mitochondria and cytoplasm were assessed using MitoSox red and dihydroethidium (DHE respectively. The product of superoxide with DHE, 2-hydroxyethidium (2-HE was acutely increased in the fiber cytosol by contractions, whereas hydroxy-MitoSox showed a slow cumulative increase. Inhibition of nitric oxide synthases increased the contraction-induced formation of hydroxy-MitoSox only with no effect on 2-HE formation. These data indicate that the acute increases in cytosolic superoxide induced by contractions are not derived from mitochondria. Data also indicate that, in muscle mitochondria, nitric oxide (NO reduces the availability of superoxide, but no effect of NO on cytosolic superoxide availability was detected. To determine the relationship of changes in superoxide to hydrogen peroxide, an alternative specific approach was used where fibers were transduced using an adeno-associated viral vector to express the hydrogen peroxide probe, HyPer within the cytoplasmic compartment. HyPer fluorescence was significantly increased in fibers following contractions, but surprisingly followed a relatively slow time course that did not appear directly related to cytosolic superoxide. These data demonstrate for the first time temporal and site specific differences in specific ROS that occur in skeletal muscle fibers during and after contractile

  7. The N-terminal region of mature mitochondrial aspartate aminotransferase can direct cytosolic dihydrofolate reductase into mitochondria in vitro.

    Science.gov (United States)

    Giannattasio, S; Azzariti, A; Marra, E; Quagliariello, E

    1994-06-30

    Two fused genes were constructed which encode for two chimeric proteins in which either 10 or 191 N-terminal amino acids of mature mitochondrial aspartate aminotransferase had been attached to the entire polypeptide chain of cytosolic dihydrofolate reductase. The precursor and mature form of mitochondrial aspartate aminotransferase, dihydrofolate reductase and both chimeric proteins were synthesized in vitro and their import into isolated mitochondria was studied. Both chimeric proteins were taken up by isolated organelles, where they became protease resistant, thus indicating the ability of the N-terminal portion of the mature moiety of the precursor of mitochondrial aspartate aminotransferase to direct cytosolic dihydrofolate reductase into mitochondria.

  8. [Cytosol malate dehydrogenase in Calicophoron ijimai trematodes and the effect of antiparasitic preparations on its activity].

    Science.gov (United States)

    Vykhrestiuk, N P; Khamatova, A Iu

    1983-01-01

    The activity and properties of malate dehydrogenase (MDH; EC 1.1.1.37) and of "malic" enzyme (EC 1.1.1.40) in cytosole of the trematode C. ijimai were determined. The activity of MDH directed to oxaloacetate formation was shown to be 14 times and maximum velocity 13 times lower than that of the reverse reaction. The apparent KM was one order higher in the direct reaction. This confirms the possibility of glycolytic pathway in C. ijimai via CO2 fixation into phosphoenolpyruvate to form oxaloacatate which is readily eliminated by active MDH. The presence of "malic" enzyme in C. ijimai testifies to the occurrence of different pathways of succinate formation in this species.

  9. Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism*

    Science.gov (United States)

    Gohil, Vishal M.; Zhu, Lin; Baker, Charli D.; Cracan, Valentin; Yaseen, Abbas; Jain, Mohit; Clish, Clary B.; Brookes, Paul S.; Bakovic, Marica; Mootha, Vamsi K.

    2013-01-01

    We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism. PMID:24142790

  10. DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

    Science.gov (United States)

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

    2016-01-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  11. Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts.

    Science.gov (United States)

    Wharton, S A; Hipkiss, A R

    1984-03-12

    Puromycyl peptides were degraded in MRC5 fibroblasts more rapidly than normal proteins labelled for the corresponding length of time for both long and short labelling periods. The degradation of the puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half-lives of normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal proteins were degraded in the lysosomes. The rapid degradation of the puromycyl peptides was not due to the inhibition of protein synthesis brought about by puromycin but was due to the structure of the substrates themselves. The degree and intracellular site of degradation of puromycyl peptides closely mimic those of abnormal (missense) proteins containing amino acid analogues.

  12. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    Thymidine kinase (TK) is a key enzyme in the salvage pathway of the nucleoside metabolism catalyzing the first phosphorylation step in TTP synthesis. Human cytosolic TK (TKl) is highly cell cycle regulated. TKl is regulated on many different levels of expression and isoforms with altered enzymatic...... properties are found in cancer cells. Investigation of these factors offers possibilities to understand the molecular background for TKl expression including to clarify general regulation patterns. It also gives valuable information for constructing new nucleoside analogs for the therapy of cancer and virus...... infections. In the first part of the present investigation a sensitive test for quantitating TKl mRNA (competitive PCR) is developed and the results show that PHA stimulated lymphocytes reveal the same pattern concerning expression of TKl mRNA and TKl enzyme activity as serum-stimulated cells. This pattern...

  13. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    Science.gov (United States)

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic reticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases. PMID:25206608

  14. Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein.

    Science.gov (United States)

    Hubert, David A; Tornero, Pablo; Belkhadir, Youssef; Krishna, Priti; Takahashi, Akira; Shirasu, Ken; Dangl, Jeffery L

    2003-11-03

    The Arabidopsis protein RPM1 activates disease resistance in response to Pseudomonas syringae proteins targeted to the inside of the host cell via the bacterial type III delivery system. We demonstrate that specific mutations in the ATP-binding domain of a single Arabidopsis cytosolic HSP90 isoform compromise RPM1 function. These mutations do not affect the function of related disease resistance proteins. RPM1 associates with HSP90 in plant cells. The Arabidopsis proteins RAR1 and SGT1 are required for the action of many R proteins, and display some structural similarity to HSP90 co-chaperones. Each associates with HSP90 in plant cells. Our data suggest that (i) RPM1 is an HSP90 client protein; and (ii) RAR1 and SGT1 may function independently as HSP90 cofactors. Dynamic interactions among these proteins can regulate RPM1 stability and function, perhaps similarly to the formation and regulation of animal steroid receptor complexes.

  15. Potassium channels-mediated electrophysiologic responses are inhibited by cytosolic phospholipase A2α ablation.

    Science.gov (United States)

    Wang, Na; Hu, Ying-Hong; Su, Li-Da

    2018-01-03

    Cytosolic phospholipase A2α (cPLA2α) is implicated in the progression of excitotoxic neuronal injury and cerebral ischemia. Previous work suggests that cPLA2α increases aberrant electrophysiologic events through attenuating K channel functions. Nevertheless, which K channels are affected by cPLA2α needs to be determined. Here we examined K channels-mediated electrophysiologic responses in hippocampal CA1 pyramidal neurons from wild-type and cPLA2α mice using simultaneous patch-clamp recording and confocal Ca imaging. After the exposure to the blockers of Ca-sensitive and A-type K channels, all CA1 neurons developed spike broadening and increased dendritic Ca transients. These effects were occluded in CA1 neurons from cPLA2α mice. Therefore, cPLA2α modulates the functions of Ca-sensitive and A-type K channels in neurotoxicity.

  16. Cytosolic fatty acid-binding proteins: subjects and tools in metabolic research

    Energy Technology Data Exchange (ETDEWEB)

    Binas, B. [Max Delbrueck Center for Molecular Medicine, Berlin-Buch (Germany)

    1998-12-31

    Fatty acid-binding proteins (FABPs) are major targets for specific binding of fatty acids in vivo. They constitute a widely expressed family of genetically related, small cytosolic proteins which very likely mediate intracellular transport of free long chain fatty acids. Genetic inhibition of FABP expression in vivo should therefore provide a useful tool to investigate and engineer fatty acid metabolism. (orig.) [Deutsch] Fettsaeurebindungsproteine (FABPs) sind wichtige Bindungsstellen fuer Fettsaeuren in vivo; sie bilden eine breit exprimierte Familie genetisch verwandter kleiner Zytosoleiweisse, die sehr wahrscheinlich den intrazellulaeren Transport unveresterter langkettiger Fettsaeuren vermitteln. Die genetische Hemmung der FABP-Expanssion in vivo bietet sich deshalb als Werkzeug zur Erforschung und gezielten Veraenderung des Fettsaeurestoffwechsels an. (orig.)

  17. Comparative inhibition of tetrameric carbonyl reductase activity in pig heart cytosol by alkyl 4-pyridyl ketones.

    Science.gov (United States)

    Shimada, Hideaki; Tanigawa, Takahiro; Matayoshi, Kazunori; Katakura, Kazufumi; Babazono, Ken; Takayama, Hiroyuki; Murahashi, Tsuyoshi; Akita, Hiroyuki; Higuchi, Toshiyuki; Eto, Masashi; Imamura, Yorishige

    2014-06-01

    The present study is to elucidate the comparative inhibition of tetrameric carbonyl reductase (TCBR) activity by alkyl 4-pyridyl ketones, and to characterize its substrate-binding domain. The inhibitory effects of alkyl 4-pyridyl ketones on the stereoselective reduction of 4-benzoylpyridine (4-BP) catalyzed by TCBR were examined in the cytosolic fraction of pig heart. Of alkyl 4-pyridyl ketones, 4-hexanoylpyridine, which has a straight-chain alkyl group of five carbon atoms, inhibited most potently TCBR activity and was a competitive inhibitor. Furthermore, cyclohexyl pentyl ketone, which is substituted by cyclohexyl group instead of phenyl group of hexanophenone, had much lower ability to be reduced than hexanophenone. These results suggest that in addition to a hydrophobic cleft corresponding to a straight-chain alkyl group of five carbon atoms, a hydrophobic pocket with affinity for an aromatic group is located in the substrate-binding domain of TCBR.

  18. Fos-Zippered GH Receptor Cytosolic Tails Act as Jak2 Substrates and Signal Transducers.

    Science.gov (United States)

    Nespital, Tobias; van der Velden, Lieke M; Mensinga, Anneloes; van der Vaart, Elisabeth D; Strous, Ger J

    2016-03-01

    Members of the Janus kinase (Jak) family initiate the majority of downstream signaling events of the cytokine receptor family. The prevailing principle is that the receptors act in dimers: 2 Jak2 molecules bind to the cytosolic tails of a cytokine receptor family member and initiate Jak-signal transducer and activator of transcription signaling upon a conformational change in the receptor complex, induced by the cognate cytokine. Due to the complexity of signaling complexes, there is a strong need for in vitro model systems. To investigate the molecular details of the Jak2 interaction with the GH receptor (GHR), we used cytosolic tails provided with leucine zippers derived from c-Fos to mimic the dimerized state of GHR. Expressed together with Jak2, fos-zippered tails, but not unzippered tails, were stabilized. In addition, the Jak-signal transducer and activator of transcription signaling pathway was activated by the fos-zippered tails. The stabilization depended also on α-helix rotation of the zippers. Fos-zippered GHR tails and Jak2, both purified from baculovirus-infected insect cells, interacted via box1 with a binding affinity of approximately 40nM. As expected, the Jak kinase inhibitor Ruxolitinib inhibited the stabilization but did not affect the c-Fos-zippered GHR tail-Jak2 interaction. Analysis by blue-native gel electrophoresis revealed high molecular-weight complexes containing both Jak2 and nonphosphorylated GHR tails, whereas Jak2-dissociated tails were highly phosphorylated and monomeric, implying that Jak2 detaches from its substrate upon phosphorylation.

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

    Science.gov (United States)

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

    2014-11-01

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

  20. Doxorubicin Regulates Autophagy Signals via Accumulation of Cytosolic Ca2+ in Human Cardiac Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Ji Hye Park

    2016-10-01

    Full Text Available Doxorubicin (DOXO is widely used to treat solid tumors. However, its clinical use is limited by side effects including serious cardiotoxicity due to cardiomyocyte damage. Resident cardiac progenitor cells (hCPCs act as key regulators of homeostasis in myocardial cells. However, little is known about the function of hCPCs in DOXO-induced cardiotoxicity. In this study, we found that DOXO-mediated hCPC toxicity is closely related to calcium-related autophagy signaling and was significantly attenuated by blocking mTOR signaling in human hCPCs. DOXO induced hCPC apoptosis with reduction of SMP30 (regucalcin and autophagosome marker LC3, as well as remarkable induction of the autophagy-related markers, Beclin-1, APG7, and P62/SQSTM1 and induction of calcium-related molecules, CaM (Calmodulin and CaMKII (Calmodulin kinase II. The results of an LC3 puncta assay further indicated that DOXO reduced autophagosome formation via accumulation of cytosolic Ca2+. Additionally, DOXO significantly induced mTOR expression in hCPCs, and inhibition of mTOR signaling by rapamycin, a specific inhibitor, rescued DOXO-mediated autophagosome depletion in hCPCs with significant reduction of DOXO-mediated cytosolic Ca2+ accumulation in hCPCs, and restored SMP30 and mTOR expression. Thus, DOXO-mediated hCPC toxicity is linked to Ca2+-related autophagy signaling, and inhibition of mTOR signaling may provide a cardio-protective effect against DOXO-mediated hCPC toxicity.

  1. A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death.

    Science.gov (United States)

    Wang, Xiaowen; Chen, Xin Jie

    2015-08-27

    Mitochondria are multifunctional organelles whose dysfunction leads to neuromuscular degeneration and ageing. The multi-functionality poses a great challenge for understanding the mechanisms by which mitochondrial dysfunction causes specific pathologies. Among the leading mitochondrial mediators of cell death are energy depletion, free radical production, defects in iron-sulfur cluster biosynthesis, the release of pro-apoptotic and non-cell-autonomous signalling molecules, and altered stress signalling. Here we identify a new pathway of mitochondria-mediated cell death in yeast. This pathway was named mitochondrial precursor over-accumulation stress (mPOS), and is characterized by aberrant accumulation of mitochondrial precursors in the cytosol. mPOS can be triggered by clinically relevant mitochondrial damage that is not limited to the core machineries of protein import. We also discover a large network of genes that suppress mPOS, by modulating ribosomal biogenesis, messenger RNA decapping, transcript-specific translation, protein chaperoning and turnover. In response to mPOS, several ribosome-associated proteins were upregulated, including Gis2 and Nog2, which promote cap-independent translation and inhibit the nuclear export of the 60S ribosomal subunit, respectively. Gis2 and Nog2 upregulation promotes cell survival, which may be part of a feedback loop that attenuates mPOS. Our data indicate that mitochondrial dysfunction contributes directly to cytosolic proteostatic stress, and provide an explanation for the association between these two hallmarks of degenerative diseases and ageing. The results are relevant to understanding diseases (for example, spinocerebellar ataxia, amyotrophic lateral sclerosis and myotonic dystrophy) that involve mutations within the anti-degenerative network.

  2. Identification and partial characterization of cytosolic progesterone-binding sites in the filamentous fungus Rhizopus nigricans.

    Science.gov (United States)

    Lenasi, H; Hudnik-Plevnik, T

    1996-06-01

    Progesterone and some other steroids have been shown to induce a steroid 11alpha-hydroxylating enzyme system requiring cytochrome P450 in the filamentous fungus Rhizopus nigricans. In the present work, we attempted to find out whether the mycelial cytosol contained progesterone-binding sites (PBS) which could function as receptors for P450-inducing steroids and might, therefore, be included in the induction process. Two types of constitutive PBS, PBS-I and PBS-II, were identified in the cytosol pretreated with dextran-coated charcoal which removed the endogenous ligand. The protein nature of these binding activities was indicated by their susceptibility to trypsin and proteinase K digestion, heat denaturation, and their resistance to DNase. Progesterone binding was rapid, the maximal level being reached after 45 min of incubation at 22 degrees C. At this temperature, dissociation of progesterone from PBS-I proceeded with a t1/2 of 17 min and that from PBS-II with a t1/2 of 133 min. The apparent Kd of PBS-I determined by Scatchard analysis was 2.1-7.0 x 10(-9)M, and Bmax 36-218 fmol/mg protein. Bmax for PBS-II was >400 fmol/mg protein, whereas the value of Kd could not be determined accurately due to the sigmoidal nature of the association kinetics. The biological role of PBS-I in transcriptional regulation is suggested by the observation that this receptor-like protein contains a functional DNA-binding domain. A specific function of PBS-I in the induction of 11alpha-hydroxylase seems to be, however, questionable because of poor correlation between the affinity and the inducing capability of corresponding steroids.

  3. Increased group IV cytosolic phospholipase A2 activity in lungs of sheep after smoke inhalation injury.

    Science.gov (United States)

    Fukuda, T; Kim, D K; Chin, M R; Hales, C A; Bonventre, J V

    1999-09-01

    Increased phospholipase A2 (PLA2) activity was measured in cytosolic fractions of lungs from sheep exposed to smoke from burning cotton or to synthetic smoke consisting of carbon and acrolein, a cotton smoke toxin. Three peaks of PLA2 activity were identified by heparin-Sepharose chromatography. The heparin-nonbinding PLA2 activity was twofold higher in the extracts from lungs exposed to smoke than in normal lungs. This activity was identified as the group IV 85-kDa cytosolic PLA2 (cPLA2). The activities of the forms of PLA2 that bound to heparin did not change after smoke exposure. Those activities showed a pH optimum of 9.0, required a millimolar Ca2+ concentration for full activity, and were inhibited by 5 mM dithiothreitol. One activity eluted at an NaCl concentration typical for group Ib and V PLA2 and had the expected substrate specificity. The other form of lung PLA2 that bound heparin was a group II PLA2. Lung myeloperoxidase activity increased progressively with increased exposure to smoke. cPLA2 was identified in sheep neutrophils. With 30 breaths of smoke exposure, there was an increase in cPLA2 activity without a difference in immunoreactivity on Western blot, indicating that the increased activity was not due to increased amounts of protein. In conclusion, smoke induces increases in resident lung cell cPLA2 activity that is likely responsible for eicosanoid production, leading to lung inflammation and bronchoconstriction.

  4. Caerulein causes translocation of protein kinase C in rat acini without increasing cytosolic free Ca2+.

    Science.gov (United States)

    Bruzzone, R; Regazzi, R; Wollheim, C B

    1988-07-01

    We investigated the relationships between changes in cytosolic free Ca2+ ([Ca2+]i) and amylase secretion in dispersed rat pancreatic acini. Although 10 pM caerulein did not raise [Ca2+]i, higher concentrations (1 nM) of the peptide elicited a prompt, marked, but transient (2-3 min) elevation of [Ca2+]i. Both concentrations of caerulein caused an almost identical release of amylase over a 30-min period. To investigate the mechanism(s) underlying Ca2+-independent secretion, we measured the effect of the secretagogue on protein kinase C activity and found that both caerulein concentrations caused a significant translocation of protein kinase C from the cytosolic to the microsomal fraction. Because 1 nM caerulein induced a greater enzyme secretion than 10 pM caerulein during the first 2-5 min of stimulation, we explored further the role of [Ca2+]i transients during the first minutes of secretion. Addition of ionomycin in the presence of 10 pM caerulein resulted in a rise in [Ca2+]i and enhanced secretion as a result of caerulein in a near additive fashion during the first 2 min of stimulation. Second, we pretreated acini for 5 min with 1 microM 12-O-tetradecanoylphorbol-13-acetate. This maneuver inhibited both caerulein-induced inositol trisphosphate formation and [Ca2+]i elevation. These findings were paralleled by a similar inhibition of caerulein-stimulated amylase release only during the first 5 min of secretion. These results indicate that 1) caerulein can stimulate amylase secretion independently of a concomitant [Ca2+]i rise, possibly by activation of protein kinase C, and 2) an elevation of [Ca2+]i serves as a trigger to enhance amylase release only during the initial phase of secretion.

  5. The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.

    Science.gov (United States)

    Patterson, Melissa A; Bandyopadhyay, Anannya; Devaraneni, Prasanna K; Woodward, Josha; Rooney, LeeAnn; Yang, Zhongying; Skach, William R

    2015-11-27

    Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. The Bimodal Lifestyle of Intracellular Salmonella in Epithelial Cells: Replication in the Cytosol Obscures Defects in Vacuolar Replication

    Science.gov (United States)

    Steele-Mortimer, Olivia

    2012-01-01

    Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens. PMID:22719929

  7. Extracellular ATP induces spikes in cytosolic free Ca(2+) but not in NADPH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B

    2011-01-01

    , we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca(2+) but, despite subsequent spikes in Ca(2+) concentration, no oscillations in oxidase activity could...

  8. THE CYTOSOLIC AND GLYCOSOMAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM TRYPANOSOMA-BRUCEI - KINETIC-PROPERTIES AND COMPARISON WITH HOMOLOGOUS ENZYMES

    NARCIS (Netherlands)

    LAMBEIR, AM; LOISEAU, AM; KUNTZ, DA; VELLIEUX, FM; MICHELS, PAM; OPPERDOES, FR

    1991-01-01

    The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like

  9. Ach1 is involved in shuttling mitochondrial acetyl units for cytosolic C2 provision in Saccharomyces cerevisiae lacking pyruvate decarboxylase

    DEFF Research Database (Denmark)

    Chen, Yun; Zhang, Yiming; Siewers, Verena

    2015-01-01

    Saccharomyces cerevisiae, acetyl-CoA is compartmentalized in the cytosol, mitochondrion, peroxisome and nucleus, and cannot be directly transported between these compartments. With the acetyl-carnitine or glyoxylate shuttle, acetyl-CoA produced in peroxisomes or the cytoplasm can be transported...

  10. Trolox-sensitive reactive oxygen species regulate mitochondrial morphology, oxidative phosphorylation and cytosolic calcium handling in healthy cells

    NARCIS (Netherlands)

    Distelmaier, F.; Valsecchi, F.; Forkink, M.; Emst-de Vries, S.E. van; Swarts, H.G.P.; Rodenburg, R.J.T.; Verwiel, E.T.P.; Smeitink, J.A.M.; Willems, P.H.G.M.; Koopman, W.J.H.

    2012-01-01

    AIMS: Cell regulation by signaling reactive oxygen species (sROS) is often incorrectly studied through extracellular oxidant addition. Here, we used the membrane-permeable antioxidant Trolox to examine the role of sROS in mitochondrial morphology, oxidative phosphorylation (OXPHOS), and cytosolic

  11. Contribution of vesicular and cytosolic dopamine to the increased striatal dopamine efflux elicited by intrastriatal injection of dexamphetamine.

    NARCIS (Netherlands)

    Watanabe, S.; Aono, Y.; Fusa, K.; Takada, K.; Saigusa, T.; Koshikawa, N.; Cools, A.R.

    2005-01-01

    Systemic administration of high doses of dexamphetamine induces a dopamine efflux that has its intracellular origin in both the vesicular, reserpine-sensitive dopamine pool and the cytosolic, alpha-methyl-para-tyrosine-sensitive, newly synthesized dopamine pool. It remains unknown whether locally

  12. Engineering acetyl coenzyme A supply : Functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Kozak, B.U.; Van Rossum, M.H.; Luttik, M.A.; Akeroyd, M.; Benjamin, K.R.; Wu, L.; De Vries, S.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.

    2014-01-01

    The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been

  13. Engineering Acetyl Coenzyme A Supply : Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Kozak, B.U.; Van Rossum, H.M.; Luttik, M.A.H.; Akeroyd, M.; Benjamin, K.R.; Wu, L.; De Vries, S.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.

    2014-01-01

    The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been

  14. The maize DWARF1 encodes a gibberellin 3-oxidase and is dual localized to the nucleus and cytosol.

    Science.gov (United States)

    Chen, Yi; Hou, Mingming; Liu, Lijuan; Wu, Shan; Shen, Yun; Ishiyama, Kanako; Kobayashi, Masatomo; McCarty, Donald R; Tan, Bao-Cai

    2014-12-01

    The maize (Zea mays) gibberellin (GA)-deficient mutant dwarf1 (d1) displays dwarfism and andromonoecy (i.e. forming anthers in the female flower). Previous characterization indicated that the d1 mutation blocked three steps in GA biosynthesis; however, the locus has not been isolated and characterized. Here, we report that D1 encodes a GA 3-oxidase catalyzing the final step of bioactive GA synthesis. Recombinant D1 is capable of converting GA20 to GA1, GA20 to GA3, GA5 to GA3, and GA9 to GA4 in vitro. These reactions are widely believed to take place in the cytosol. However, both in vivo GFP fusion analysis and western-blot analysis of organelle fractions using a D1-specific antibody revealed that the D1 protein is dual localized in the nucleus and cytosol. Furthermore, the upstream gibberellin 20-oxidase1 (ZmGA20ox1) protein was found dual localized in the nucleus and cytosol as well. These results indicate that bioactive GA can be synthesized in the cytosol and the nucleus, two compartments where GA receptor Gibberellin-insensitive dwarf protein1 exists. Furthermore, the D1 protein was found to be specifically expressed in the stamen primordia in the female floret, suggesting that the suppression of stamen development is mediated by locally synthesized GAs. © 2014 American Society of Plant Biologists. All Rights Reserved.

  15. Differential changes in the activity of cytosolic and vacuolar trehalases along the growth cycle of Saccharomyces cerevisiae.

    Science.gov (United States)

    San Miguel, P F; Argüelles, J C

    1994-07-06

    Saccharomyces cerevisiae cells contain two intracellular and soluble trehalases with distinct subcellular location (cytosol and vacuoles, respectively). Both enzymes showed an opposite pattern of activity along the growth cycle. Activity of the cytosolic trehalase was high in cells growing exponentially on fermentable sugars (glucose, mannose or galactose) and sharply decayed as the cultures enter stationary phase coinciding with the beginning of trehalose biosynthesis. By contrast, vacuolar trehalase was only detectable in glucose-grown resting cells or in cultures growing on respiratory substrates (glycerol or ethanol). This enzyme was partially derepressed in the mutant hex2, which is deficient in glucose repression. Addition of fresh YPD medium to stationary-phase cultures induced the sudden reactivation of cytosolic trehalase with the concomitant slower inactivation of vacuolar trehalase. However, addition of glucose or various nitrogen sources alone had only a minor effect on both activities. The presence of cycloheximide had no effect on cytosolic trehalase, whereas completely blocked the appearance of vacuolar trehalase suggesting the requirement of protein synthesis 'de novo'.

  16. Oestrogen receptors in endometrial cytosol of gilts on days 10-13 of oestrous cycle and pregnancy.

    NARCIS (Netherlands)

    Meulen, van der J.; Helmond, F.A.; Oudenaarden, C.P.J.

    1994-01-01

    Cytosolic oestrogen receptors (ER(c)) were determined with a Dextran-coated charcoal assay and Scatchard plot analysis in endometrial tissue of non-pregnant and pregnant gilts on Days 10-13 after standing oestrus. The Kd of the ER(c) was 0.40 /- 0.04 nM (mean /- SEM) and was not affected by day or

  17. Alternative translational initiation of ATP sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Anne-Sophie eBohrer

    2015-01-01

    Full Text Available Plants assimilate inorganic sulfate into sulfur-containing vital metabolites. ATP sulfurylase (ATPS is the enzyme catalyzing the key entry step of the sulfate assimilation pathway in both plastids and cytosol in plants. Arabidopsis thaliana has four ATPS genes (ATPS1, -2, -3 and -4 encoding ATPS pre-proteins containing N-terminal transit peptide sequences for plastid targeting, however, the genetic identity of the cytosolic ATPS has remained unverified. Here we show that Arabidopsis ATPS2 dually encodes plastidic and cytosolic ATPS isoforms, differentiating their subcellular localizations by initiating translation at AUGMet1 to produce plastid-targeted ATPS2 pre-proteins or at AUGMet52 or AUGMet58 within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUGMet52 or AUGMet58 was verified by expressing a tandem-fused synthetic gene, ATPS2(5’UTR-His12:Renilla luciferase:ATPS2(Ile13-Val77:firefly luciferase, under a single constitutively active CaMV 35S promoter in Arabidopsis protoplasts and examining the activities of two different luciferases translated in-frame with split N-terminal portions of ATPS2. Introducing missense mutations at AUGMet52 and AUGMet58 significantly reduced the firefly luciferase activity, while AUGMet52 was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUGMet52 or AUGMet58 was not modulated by changes in sulfate conditions. The dual localizations of ATPS2 in plastids and cytosol were further evidenced by expression of ATPS2-GFP fusion proteins in Arabidopsis protoplasts and transgenic lines, while they were also under control of tissue-specific ATPS2 promoter activity found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots.

  18. Faster and stronger manifestation of mitochondrial diseases in skeletal muscle than in heart related to cytosolic inorganic phosphate (Pi) accumulation

    Science.gov (United States)

    2016-01-01

    A model of the cell bioenergetic system was used to compare the effect of oxidative phosphorylation (OXPHOS) deficiencies in a broad range of moderate ATP demand in skeletal muscle and heart. Computer simulations revealed that kinetic properties of the system are similar in both cases despite the much higher mitochondria content and “basic” OXPHOS activity in heart than in skeletal muscle, because of a much higher each-step activation (ESA) of OXPHOS in skeletal muscle than in heart. Large OXPHOS deficiencies lead in both tissues to a significant decrease in oxygen consumption (V̇o2) and phosphocreatine (PCr) and increase in cytosolic ADP, Pi, and H+. The main difference between skeletal muscle and heart is a much higher cytosolic Pi concentration in healthy tissue and much higher cytosolic Pi accumulation (level) at low OXPHOS activities in the former, caused by a higher PCr level in healthy tissue (and higher total phosphate pool) and smaller Pi redistribution between cytosol and mitochondria at OXPHOS deficiency. This difference does not depend on ATP demand in a broad range. A much greater Pi increase and PCr decrease during rest-to-moderate work transition in skeletal muscle at OXPHOS deficiencies than at normal OXPHOS activity significantly slows down the V̇o2 on-kinetics. Because high cytosolic Pi concentrations cause fatigue in skeletal muscle and can compromise force generation in skeletal muscle and heart, this system property can contribute to the faster and stronger manifestation of mitochondrial diseases in skeletal muscle than in heart. Shortly, skeletal muscle with large OXPHOS deficiencies becomes fatigued already during low/moderate exercise. PMID:27283913

  19. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    Directory of Open Access Journals (Sweden)

    Roger Huerlimann

    Full Text Available The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT, and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid and in two forms (homomeric and heteromeric. All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa and Chromista (Stramenopiles, Haptophyta and Cryptophyta have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO, Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta. These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was

  20. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    KAUST Repository

    Huerlimann, Roger

    2015-07-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  1. Tyrosylprotein sulfotransferase-1 and tyrosine sulfation of chemokine receptor 4 are induced by Epstein-Barr virus encoded latent membrane protein 1 and associated with the metastatic potential of human nasopharyngeal carcinoma.

    Directory of Open Access Journals (Sweden)

    Juan Xu

    Full Text Available The latent membrane protein 1 (LMP1, which is encoded by the Epstein-Barr virus (EBV, is an important oncogenic protein that is closely related to carcinogenesis and metastasis of nasopharyngeal carcinoma (NPC, a prevalent cancer in China. We previously reported that the expression of the functional chemokine receptor CXCR4 is associated with human NPC metastasis. In this study, we show that LMP1 induces tyrosine sulfation of CXCR4 through tyrosylprotein sulfotransferase-1 (TPST-1, an enzyme that is responsible for catalysis of tyrosine sulfation in vivo, which is likely to contribute to the highly metastatic character of NPC. LMP1 could induce tyrosine sulfation of CXCR4 and its associated cell motility and invasiveness in a NPC cell culture model. In contrast, the expression of TPST-1 small interfering RNA reversed LMP1-induced tyrosine sulfation of CXCR4. LMP1 conveys signals through the epidermal growth factor receptor (EGFR pathway, and EGFR-targeted siRNA inhibited the induction of TPST-1 by LMP1. We used a ChIP assay to show that EGFR could bind to the TPST-1 promoter in vivo under the control of LMP1. A reporter gene assay indicated that the activity of the TPST-1 promoter could be suppressed by deleting the binding site between EGFR and TPST-1. Finally, in human NPC tissues, the expression of TPST-1 and LMP1 was directly correlated and clinically, the expression of TPST-1 was associated with metastasis. These results suggest the up-regulation of TPST-1 and tyrosine sulfation of CXCR4 by LMP1 might be a potential mechanism contributing to NPC metastasis.

  2. Modulation of adenylate cyclase activity by a cytosolic factor following chronic opiate exposure in neuroblastoma x glioma NG108-15 hybrid cells.

    Science.gov (United States)

    Griffin, M T; Law, P Y; Loh, H H

    1983-01-01

    A soluble cytosolic factor from neuroblastoma x glioma NG108-15 hybrid cells stimulates adenylate cyclase activity in isolated membrane preparations. This cytosolic component is heat stable, pronase insensitive, has a molecular weight less than 350 daltons and an absorbance peak at 260 nm. The stimulation is immediate, independent of Ca++ and exhibits a sigmoidal concentration dependency curve. The cytosolic factor stimulated adenylate cyclase activity in etorphine treated cells (100 nM etorphine, 16 hrs) to a greater extent than in control cells. In addition, cytosolic factor derived from etorphine treated cells, as compared to control cells, displayed an increased capacity to stimulate adenylate cyclase. It is suggested that the observed cytosolic factor may be adenosine and that cells chronically treated with an opiate exhibit an increase in both concentration and sensitivity to this agent.

  3. Interplay Between Cytosolic Free Zn2+ and Mitochondrion Morphological Changes in Rat Ventricular Cardiomyocytes.

    Science.gov (United States)

    Billur, Deniz; Tuncay, Erkan; Okatan, Esma Nur; Olgar, Yusuf; Durak, Aysegul Toy; Degirmenci, Sinan; Can, Belgin; Turan, Belma

    2016-11-01

    The Zn2+ in cardiomyocytes is buffered by structures near T-tubulus and/or sarcoplasmic/endoplasmic reticulum (S(E)R) while playing roles as either an antioxidant or a toxic agent, depending on the concentration. Therefore, we aimed first to examine a direct effect of ZnPO4 (extracellular exposure) or Zn2+ pyrithione (ZnPT) (intracellular exposure) application on the structure of the mitochondrion in ventricular cardiomyocytes by using histological investigations. The light microscopy data demonstrated that Zn2+ exposure induced marked increases on cellular surface area, an indication of hypertrophy, in a concentration-dependent manner. Furthermore, a whole-cell patch-clamp measurement of cell capacitance also supported the hypertrophy in the cells. We observed marked increases in mitochondrial matrix/cristae area and matrix volume together with increased lysosome numbers in ZnPO4- or ZnPT-incubated cells by using transmission electron microscopy, again in a concentration-dependent manner. Furthermore, we observed notable clustering and vacuolated mitochondrion, markedly disrupted and damaged myofibrils, and electron-dense small granules in Zn2+-exposed cells together with some implications of fission-fusion defects in the mitochondria. Moreover, we observed marked depolarization in mitochondrial membrane potential during 1-μM ZnPT minute applications by using confocal microscopy. We also showed that 1-μM ZnPT incubation induced significant increases in the phosphorylation levels of GSK3β (Ser21 and Ser9), Akt (Ser473), and NFκB (Ser276 and Thr254) together with increased expression levels in ER stress proteins such as GRP78 and calregulin. Furthermore, a new key player at ER-mitochondria sites, promyelocytic leukemia protein (PML) level, was markedly increased in ZnPT-incubated cells. As a summary, our present data suggest that increased cytosolic free Zn2+ can induce marked alterations in mitochondrion morphology as well as depolarization in mitochondrion

  4. Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

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    Sasnauskas Kęstutis

    2011-05-01

    Full Text Available Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN and measles hemagglutinin (MeH in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A and is closely associated with small heat shock proteins (sHsps that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of

  5. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

    Science.gov (United States)

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-10-28

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

  6. Evasion of innate cytosolic DNA sensing by a gammaherpesvirus facilitates establishment of latent infection1

    Science.gov (United States)

    Sun, Chenglong; Schattgen, Stefan A.; Pisitkun, Prapaporn; Jorgensen, Joan P.; Hilterbrand, Adam T.; Wang, Lucas J.; West, John A.; Hansen, Kathrine; Horan, Kristy A.; Jakobsen, Martin R.; O'Hare, Peter; Adler, Heiko; Sun, Ren; Ploegh, Hidde L.; Damania, Blossom; Upton, Jason W.; Fitzgerald, Katherine A.; Paludan, Søren R.

    2014-01-01

    Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is currently limited knowledge on viruses targeting the innate DNA sensing pathway and also on how the innate system impacts on the latent reservoir of herpesvirus infections. Here we report that murine gammaherpesvirus MHV68, in contrast to alpha- and beta-herpesviruses, induce very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA sensing pathways. Interestingly, a MHV68 mutant lacking the deubiquitinase (DUB) activity, embedded within the large tegument protein ORF64, gained the capacity to stimulate the DNA-activated STING pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and AIM2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which instead localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild type but not STING-deficient mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA sensing pathway, which in the MHV68 model facilitates establishment of infections. PMID:25595793

  7. Cytosolic invertase contributes to the supply of substrate for cellulose biosynthesis in developing wood.

    Science.gov (United States)

    Rende, Umut; Wang, Wei; Gandla, Madhavi Latha; Jönsson, Leif J; Niittylä, Totte

    2017-04-01

    Carbon for cellulose biosynthesis is derived from sucrose. Cellulose is synthesized from uridine 5'-diphosphoglucose (UDP-glucose), but the enzyme(s) responsible for the initial sucrose cleavage and the source of UDP-glucose for cellulose biosynthesis in developing wood have not been defined. We investigated the role of CYTOSOLIC INVERTASEs (CINs) during wood formation in hybrid aspen (Populus tremula × tremuloides) and characterized transgenic lines with reduced CIN activity during secondary cell wall biosynthesis. Suppression of CIN activity by 38-55% led to a 9-13% reduction in crystalline cellulose. The changes in cellulose were reflected in reduced diameter of acid-insoluble cellulose microfibrils and increased glucose release from wood upon enzymatic digestion of cellulose. Reduced CIN activity decreased the amount of the cellulose biosynthesis precursor UDP-glucose in developing wood, pointing to the likely cause of the cellulose phenotype. The findings suggest that CIN activity has an important role in the cellulose biosynthesis of trees, and indicate that cellulose biosynthesis in wood relies on a quantifiable UDP-glucose pool. The results also introduce a concept of altering cellulose microfibril properties by modifying substrate supply to cellulose biosynthesis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  8. Fluid flow modulates vascular endothelial cytosolic calcium responses to adenine nucleotides.

    Science.gov (United States)

    Shen, J; Luscinskas, F W; Gimbrone, M A; Dewey, C F

    1994-04-01

    To determine whether fluid flow influences the action of soluble vasoactive agonists on vascular endothelium. Confluent monolayers of bovine aortic endothelial cells (BAEC) were cultured on glass coverslips, prelabeled with the Ca(2+)-sensitive dye fura-2, and placed in a parallel-plate flow chamber designed to generate defined laminar fluid flow. Cytosolic free Ca2+ concentration ([Ca2+]i) in individual BAEC was monitored during perfusion with medium containing adenine nucleotide under defined flow conditions. Continuous perfusion with ATP (0.3-3.0 microM) or ADP (0.1-1.0 microM) evoked repetitive oscillations in [Ca2+]i in individual BAEC. The frequency of the [Ca2+]i oscillations was dependent on both nucleotide concentration and levels of applied shear stress; at constant bulk concentration of nucleotide, the frequency increased with shear stress. Stopping flow in the continuous presence of agonists immediately extinguished the oscillatory response. Elimination of extracellular Ca2+ did not inhibit the [Ca2+]i oscillations. In the presence of nonhydrolyzable nucleotide analog, ATP gamma S or ADP beta S, application of flow resulted in similar shear-dependent [Ca2+]i oscillations, suggesting that flow modulation of the [Ca2+]i response was not simply due to depletion of ATP or ADP in the vicinity of BAEC monolayers as a result of hydrolysis of nucleotides by ectonucleotidases. These findings suggest that local hemodynamic conditions may modulate the action of vasoactive agents on the vascular endothelium in vivo.

  9. [Isolation of tropomyosin particles from the cytosol of cultured cells and their protein composition analysis].

    Science.gov (United States)

    Bobkov, D E; Aĭzenshtadt, A A; Kropacheva, I V; Pinaev, G P

    2012-01-01

    The presence of actin-binding protein, tropomyosin, shaped as particles or protein complexes that have no bonds with actin structures were found while the analisys of structural rearrangements of actin cytoskeleton. However, their functioning is still unknown. To study the composition and properties of these protein complexes a novel method of their separation from the cells without destroying the structures of the cytoskeleton have been developed. The protein composition of isolated tropomyosin particles has been analised by gel filtration, electrophoresis and Western blotting. They appeared to be a multimolecular complexes of about 700 kDa. Beside the tropomyosin and actin these complexes also contain the Hsp70, Hsp90 and myosin-9 identified by mass spectrometry analisys. Also, under inhibition of deacetylases by trichostatin A, changes in the number of particles and redistribution of tropomyosin between cytosol and cytoskeleton take place along with actin cytoskeleton rearrangements. The results obtained give a reason to assume that these multimolecular complexes may participate in the process of reorganization of the actin microfilaments.

  10. Cytosolic Ca(2+) as a multifunctional modulator is required for spermiogenesis in Ascaris suum.

    Science.gov (United States)

    Shang, Yunlong; Chen, Lianwan; Liu, Zhiyu; Wang, Xia; Ma, Xuan; Miao, Long

    2013-06-01

    The dynamic polar polymers actin filaments and microtubules are usually employed to provide the structural basis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the ability to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly understood. Here we show that Ca(2+) oscillations induced by the Ca(2+) release from intracellular Ca(2+) store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation. The chelation of cytosolic Ca(2+) suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing exogenous Ca(2+) into sperm cells. Ca(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca(2+) promotes MSP disassembly by activating Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase calcineurin. In addition, Ca(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane, a regulated exocytosis required for sperm motility. Thus, Ca(2+) plays multifunctional roles during sperm activation in Ascaris suum.

  11. Plasma Albumin Induces Cytosolic Calcium Oscilations and DNA Synthesis in Human Cultured Astrocytes

    Directory of Open Access Journals (Sweden)

    Lorena Vega-Zelaya

    2014-01-01

    Full Text Available So far, a little is known about transition from normal to focal epileptic brain, although disruption in blood-brain barrier and albumin had recently involved. The main objective of this work is to characterize the response of cultured human astrocytes to plasma albumin, including induction of DNA synthesis. Cortical tissue was obtained from 9 patients operated from temporal lobe epilepsy. Astrocytes were cultured for 3-4 weeks and cytosolic calcium concentration (Ca2+c was measured. Bovine and human plasma albumin were used. We observed that low albumin concentration decreases Ca2+c, while higher concentration, induces increase in Ca2+c. It was shown that increase in Ca2+c was mediated by inositol 1,4,5-trisphosphate and released from internal stores. Increase in Ca2+c was reduced to 19% by blocking the transforming growth factor-beta (TGF-βR receptor. Albumin induces DNA synthesis in a dose-response manner. Finally, induction of DNA synthesis can be partially blocked by heparin and block of TGF-β; however, the combination of both incompletely inhibits DNA synthesis. Therefore, results suggest that mechanisms other than Ca2+ signals and TGF-β receptor activation might induce DNA synthesis in a lesser degree. These results may be important to further understand the mechanisms involved in the transition from normal to focal epileptic brain.

  12. Agonist-selective effects of opioid receptor ligands on cytosolic calcium concentration in rat striatal neurons.

    Science.gov (United States)

    Brailoiu, G Cristina; Deliu, Elena; Hooper, Robert; Dun, Nae J; Undieh, Ashiwel S; Adler, Martin W; Benamar, Khalid; Brailoiu, Eugen

    2012-06-01

    Buprenorphine is an opioid receptor ligand whose mechanism of action is incompletely understood. Using Ca(2+) imaging, we assessed the effects of buprenorphine, β-endorphin, and morphine on cytosolic Ca(2+) concentration [Ca(2+)](i), in rat striatal neurons. Buprenorphine (0.01-1 μM) increased [Ca(2+)](i) in a dose-dependent manner in a subpopulation of rat striatal neurons. The effect of buprenorphine was largely reduced by naloxone, a non-selective opioid receptor antagonist, but not by μ, κ, δ or NOP-selective antagonists. β-Endorphin (0.1 μM) increased [Ca(2+)](i) with a lower amplitude and slower time course than buprenorphine. Similar to buprenorphine, the effect of β-endorphin was markedly decreased by naloxone, but not by opioid-selective antagonists. Morphine (0.1-10 μM), did not affect [Ca(2+)](i) in striatal neurons. Our results suggest that buprenorphine and β-endorphin act on a distinct type/subtype of plasmalemmal opioid receptors or activate intracellular opioid-like receptor(s) in rat striatal neurons. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  13. Identification of Short Hydrophobic Cell-Penetrating Peptides for Cytosolic Peptide Delivery by Rational Design.

    Science.gov (United States)

    Schmidt, Samuel; Adjobo-Hermans, Merel J W; Kohze, Robin; Enderle, Thilo; Brock, Roland; Milletti, Francesca

    2017-02-15

    Cell-penetrating peptides (CPPs) enhance the cellular uptake of membrane-impermeable molecules. Most CPPs are highly cationic, potentially increasing the risk of toxic side effects and leading to accumulation in organs such as the liver. As a consequence, there is an unmet need for less cationic CPPs. However, design principles for effective CPPs are still missing. Here, we demonstrate a design principle based on a classification of peptides according to accumulated side-chain polarity and hydrophobicity. We show that in comparison to randomly selected peptides, CPPs cover a distinct parameter space. We designed peptides of only six to nine amino acids with a maximum of three positive charges covering this property space. All peptides were tested for cellular uptake and subcellular distribution. Following an initial round of screening we enriched the collection with short and hydrophobic peptides and introduced d-amino acid substitutions and lactam bridges which increased cell uptake, in particular for long-term incubation. Using a GFP complementation assay, for the most active peptides we demonstrate cytosolic delivery of a biologically active cargo peptide.

  14. Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

    Directory of Open Access Journals (Sweden)

    Martino L. Di Salvo

    2013-01-01

    Full Text Available Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP- dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stable gem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and the ε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.

  15. A Model-Independent Algorithm to Derive Ca2+ Fluxes Underlying Local Cytosolic Ca2+ Transients

    Science.gov (United States)

    Ventura, Alejandra C.; Bruno, Luciana; Demuro, Angelo; Parker, Ian; Ponce Dawson, Silvina

    2005-01-01

    Local intracellular Ca2+ signals result from Ca2+ flux into the cytosol through individual channels or clusters of channels. To gain a mechanistic understanding of these events we need to know the magnitude and spatial distribution of the underlying Ca2+ flux. However, this is difficult to infer from fluorescence Ca2+ images because the distribution of Ca2+-bound dye is affected by poorly characterized processes including diffusion of Ca2+ ions, their binding to mobile and immobile buffers, and sequestration by Ca2+ pumps. Several methods have previously been proposed to derive Ca2+ flux from fluorescence images, but all require explicit knowledge or assumptions regarding these processes. We now present a novel algorithm that requires few assumptions and is largely model-independent. By testing the algorithm with both numerically generated image data and experimental images of sparklets resulting from Ca2+ flux through individual voltage-gated channels, we show that it satisfactorily reconstructs the magnitude and time course of the underlying Ca2+ currents. PMID:15681645

  16. Free cytosolic calcium and secretagogue-stimulated initial pancreatic exocrine secretion.

    Science.gov (United States)

    Krims, P E; Pandol, S J

    1988-01-01

    In order to establish the role of secretagogue-induced changes in free cytosolic Ca2+ ([Ca2+]i) in pancreatic enzyme secretion, we measured the effects of carbachol, cholecystokinin-octapeptide (CCK-OP), bombesin, substance P, and bromo-A23187 on amylase release and [Ca2+]i in guinea pig pancreatic acini loaded with the Ca2+-selective fluorescent indicator, fura-2. Evaluation of time courses and dose-response curves indicated that carbachol, CCK-OP, bombesin, and substance P cause extracellular Ca2+-independent transient increases in [Ca2+]i and transient bursts in amylase release (initial secretion). The potencies for the secretagogues to increase [Ca2+]i and initial amylase release were similar. Bromo-A23187 also caused an extracellular Ca2+-independent transient increase in [Ca2+]i and amylase release. In the absence of extracellular Ca2+, sequential additions of substance P followed by carbachol caused transient increases in [Ca2+]i correlating with transient bursts in amylase release. In contrast, in acini first treated with carbachol, the ability of substance P to increase [Ca2+]i and amylase release was blocked. Sustained secretion caused by the secretagogues was dependent on extracellular Ca2+ but occurred at basal [Ca2+]i. Increasing [Ca2+]i during the sustained phase of stimulation by increasing the extracellular Ca2+ concentration or with bromo-A23187 did not increase the rate of sustained secretion.

  17. Circadian waves of cytosolic calcium concentration and long-range network connections in rat suprachiasmatic nucleus.

    Science.gov (United States)

    Hong, Jin Hee; Jeong, Byeongha; Min, Cheol Hong; Lee, Kyoung J

    2012-05-01

    The suprachiasmatic nucleus (SCN) is the master clock in mammals governing the daily physiological and behavioral rhythms. It is composed of thousands of clock cells with their own intrinsic periods varying over a wide range (20-28 h). Despite this heterogeneity, an intact SCN maintains a coherent 24 h periodic rhythm through some cell-to-cell coupling mechanisms. This study examined how the clock cells are connected to each other and how their phases are organized in space by monitoring the cytosolic free calcium ion concentration ([Ca(2+)](c)) of clock cells using the calcium-binding fluorescent protein, cameleon. Extensive analysis of 18 different organotypic slice cultures of the SCN showed that the SCN calcium dynamics is coordinated by phase-synchronizing networks of long-range neurites as well as by diffusively propagating phase waves. The networks appear quite extensive and far-reaching, and the clock cells connected by them exhibit heterogeneous responses in their amplitudes and periods of oscillation to tetrodotoxin treatments. Taken together, our study suggests that the network of long-range cellular connectivity has an important role for the SCN in achieving its phase and period coherence. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  18. Amino acid starvation has opposite effects on mitochondrial and cytosolic protein synthesis.

    Directory of Open Access Journals (Sweden)

    Mark A Johnson

    Full Text Available Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.

  19. Amino Acid Starvation Has Opposite Effects on Mitochondrial and Cytosolic Protein Synthesis

    Science.gov (United States)

    Pearce, Sarah F.; Rorbach, Joanna; He, Jiuya; Brea-Calvo, Gloria; Minczuk, Michal; Reyes, Aurelio; Holt, Ian J.; Spinazzola, Antonella

    2014-01-01

    Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation. PMID:24718614

  20. Enhanced proteomic analysis of Streptomyces peucetius cytosolic protein using optimized protein solubilization protocol.

    Science.gov (United States)

    Lee, Kwang Won; Song, Eunjung; Kim, June-Hyung; Lee, Hei Chan; Liou, Kwang Kyoung; Sohng, Jae Kyung; Kim, Byung-Gee

    2007-01-01

    Improvements in the dissolution of proteins in two-dimensional gel electrophoresis have greatly advanced the ability to analyze the proteomes of microorganisms under a wide variety of physiological conditions. This study examined the effect of various combinations of chaotropic agents, a reducing agent, and a detergent on the dissolution of the Streptomyces peucetius cytosolic proteins. The use of urea alone in a rehydration buffer as a chaotropic agent gave the proteome a higher solubility than any of the urea and thiourea combinations, and produced the highest resolution and clearest background in two-dimensional gel electrophoresis. Two % CHAPS, as a detergent in a rehydration buffer, improved the protein solubility. After examining the effect of several concentrations of reducing agent, 50 mM DTT in a rehydration buffer was found to be an optimal condition for the proteome analysis of Streptomyces. Using this optimized buffer condition, more than 2,000 distinct and differentially expressed soluble proteins could be resolved using two-dimensional gel electrophoresis with a pI ranging from 4-7. Under this optimized condition, 15 novel small proteins with low-level expression, which could not be analyzed under the non-optimized conditions, were identified. Overall, the optimized condition helped produce a better reference gel for Streptomyces peucetius.

  1. Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport

    Science.gov (United States)

    Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2013-01-01

    Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment. PMID:23350036

  2. New insights into the posttranslational regulation of human cytosolic thioredoxin by S-palmitoylation

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Zhiyu; Zhong, Liangwei, E-mail: liazho@ucas.ac.cn

    2015-05-15

    High level of palmitate is associated with metabolic disorders. We recently showed that enhanced level of S-palmitoylated cytosolic thioredoxin (Trx1) in mouse liver was new characteristic feature of insulin resistance. However, our understanding of the effect of S-palmitoylation on Trx1 is limited, and the tissue specificity of Trx1 S-palmitoylation is unclear. Here we show that S-palmitoylation also occurs at Cys73 of Trx1 in living endothelial cells, and the level of S-palmitoylated Trx1 undergoes regulation by insulin signaling. Trx1 prefers thiol-thioester exchange with palmitoyl-CoA to acetyl-CoA. S-palmitoylation alters conformation or secondary structure of Trx1, as well as decreases the ability of Trx1 to transfer electrons from thioredoxin reductase to S-nitrosylated protein–tyrosine phosphatase 1B and S-nitroso-glutathione. Our results demonstrate that S-palmitoylation is an important post-translational modification of human Trx1. - Highlights: • S-palmitoylation occurs at Cys73 of Trx1 in living endothelial cells. • Insulin signaling may regulate level of S-palmitoylated Trx1 in the cells. • S-palmitoylation plays significant effects on Trx1 structure and functions.

  3. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Klein, Michael G.; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching (Takeda Cali)

    2016-07-01

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

  4. Cytosolic phospholipase A{sub 2} gene in human and rat: Chromosomal localization and polymorphic markers

    Energy Technology Data Exchange (ETDEWEB)

    Tay, A.; Simon, J.S.; Jacob, H.J. [Univ. of Toronto (Canada)] [and others

    1995-03-01

    The authors report the chromosomal localization and a simple sequence repeat (SSR) in the cytosolic phospholipase A{sub 2} (cPLA{sub 2}) gene in both human and rat. A (CA){sub 18} repeat in the promoter of the rat gene was determined to exhibit length polymorphism when analyzed using the polymerase chain reaction (PCR) in 19 different inbred rat strains. Genotyping for this marker in 234 F{sub 2} progeny of a SHRXBN intercross mapped the gene to rat chromosome 13. Using a PCR strategy, a fragment of the promoter for the human gene was isolated, and a (CA){sub 18} repeat was identified. Since this marker displayed a low heterozygosity index, they also identified a mononucleotide repeat in the promoter for cPLA{sub 2} that displayed a polymorphism information content value of 0.76. The human gene was mapped using fluorescence in situ hybridization (FISH) to chromosome 1q25. Of interest, the gene encoding the enzyme prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2), which acts on the arachidonic acid product of cPLA{sub 2}, was previously localized to this same chromosomal region, raising the possibility of coordinate regulation. Identification of intragenic markers may facilitate studies of polymorphic variants of these genes as candidates for disorders in which perturbations of the eicosanoid cascade may play a role. 20 refs., 3 figs., 2 tabs.

  5. Nanoliposomal delivery of cytosolic phospholipase A2 inhibitor arachidonyl trimethyl ketone for melanoma treatment.

    Science.gov (United States)

    Gowda, Raghavendra; Dinavahi, Saketh S; Iyer, Soumya; Banerjee, Shubhadeep; Neves, Rogerio I; Pameijer, Colette R; Robertson, Gavin P

    2018-01-06

    Drug resistance and toxicity are major limitations of cancer treatment and frequently occurs in melanoma therapy. Nanotechnology can decrease drug resistance by improving drug delivery, with limited toxicity. This study details the development of nanoparticles containing arachidonyl trifluoromethyl ketone (ATK), a cytosolic phospholipase A2 inhibitor, which can inhibit multiple key pathways responsible for the development of recurrent resistant disease. Free ATK is toxic, limiting its efficacy as a therapeutic agent. Hence, a novel nanoliposomal delivery system called NanoATK was developed, which loads 61.7% of the compound and was stable at 4oC for 12 weeks. The formulation decreased toxicity-enabling administration of higher doses, which was more effective at killing melanoma cells compared to free-ATK. Mechanistically, NanoATK decreased cellular proliferation and triggered apoptosis to inhibit melanoma xenograft tumor growth without affecting animal weight. Functionally, it inhibited the cPLA2, AKT, and STAT3 pathways. Our results suggest the successful preclinical development of a unique nanoliposomal formulation containing ATK for the treatment of melanoma. Copyright © 2018. Published by Elsevier Inc.

  6. Targeting Cytosolic Phospholipase A2α for Novel Anti-Inflammatory Agents.

    Science.gov (United States)

    Soubhye, Jalal; van Antwerpen, Pierre; Dufrasne, Francois

    2018-01-16

    Group IV cytosolic phospholipase A2 (cPLA2α) plays a critical role in inflammatory processes. It produces arachidonic acid which is the main source of the pro-inflammatory eicosanoids mediators that are important in innate immune system. In some cases, these pro-inflammatory mediators cause damages to the host tissues and therefore promote autoimmune diseases. Consequently, development of potent inhibitors against cPLA2α could improve the therapy of inflammatory diseases. In the last two decades, intense efforts have been done to find potent cPLA2α inhibitors. Several scaffolds have been developed with the use of structure-activity relationship (SAR) studies, and potent inhibitors have been obtained. The poor absorption of these compounds from intestine was the main challenge for clinical application. This review illustrates the search for cPLA2α inhibitors, their SAR studies and biological effects. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. Holocarboxylase Synthetase: A Moonlighting Transcriptional Coregulator of Gene Expression and a Cytosolic Regulator of Biotin Utilization.

    Science.gov (United States)

    León-Del-Río, Alfonso; Valadez-Graham, Viviana; Gravel, Roy A

    2017-08-21

    The vitamin biotin is an essential nutrient for the metabolism and survival of all organisms owing to its function as a cofactor of enzymes collectively known as biotin-dependent carboxylases. These enzymes use covalently attached biotin as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In human cells, biotin-dependent carboxylases catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is attached to apocarboxylases by a biotin ligase: holocarboxylase synthetase (HCS) in mammalian cells and BirA in microbes. Despite their evolutionary distance, these proteins share structural and sequence similarities, underscoring their importance across all life forms. However, beyond its role in metabolism, HCS participates in the regulation of biotin utilization and acts as a nuclear transcriptional coregulator of gene expression. In this review, we discuss the function of HCS and biotin in metabolism and human disease, a putative role for the enzyme in histone biotinylation, and its participation as a nuclear factor in chromatin dynamics. We suggest that HCS be classified as a moonlighting protein, with two biotin-dependent cytosolic metabolic roles and a distinct biotin-independent nuclear coregulatory function.

  8. Cytosolic Phospholipase A2 Protein as a Novel Therapeutic Target for Spinal Cord Injury

    Science.gov (United States)

    Liu, Nai-Kui; Deng, Ling-Xiao; Zhang, Yi Ping; Lu, Qing-Bo; Wang, Xiao-Fei; Hu, Jian-Guo; Oakes, Eddie; Bonventre, Joseph V; Shields, Christopher B; Xu, Xiao-Ming

    2014-01-01

    Objective The objective of this study was to investigate whether cytosolic phospholipase A2 (cPLA2), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of spinal cord injury (SCI). Methods A combination of molecular, histological, immunohistochemical, and behavioral assessments were used to test whether blocking cPLA2 activation pharmacologically or genetically reduced cell death, protected spinal cord tissue, and improved behavioral recovery after a contusive SCI performed at the 10th thoracic level in adult mice. Results SCI significantly increased cPLA2 expression and activation. Activated cPLA2 was localized mainly in neurons and oligodendrocytes. Notably, the SCI-induced cPLA2 activation was mediated by the extracellular signal-regulated kinase signaling pathway. In vitro, activation of cPLA2 by ceramide-1-phosphate or A23187 induced spinal neuronal death, which was substantially reversed by arachidonyl trifluoromethyl ketone, a cPLA2 inhibitor. Remarkably, blocking cPLA2 pharmacologically at 30 minutes postinjury or genetically deleting cPLA2 in mice ameliorated motor deficits, and reduced cell loss and tissue damage after SCI. Interpretation cPLA2 may play a key role in the pathogenesis of SCI, at least in the C57BL/6 mouse, and as such could be an attractive therapeutic target for ameliorating secondary tissue damage and promoting recovery of function after SCI. PMID:24623140

  9. Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo

    Science.gov (United States)

    Rossano, Adam J; Chouhan, Amit K; Macleod, Gregory T

    2013-01-01

    All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pHcyto) shifts are known to accompany nerve activity in situ, but technological limitations have prevented characterization of such shifts in vivo. Genetically encoded pH-indicators (GEpHIs) allow for tissue-specific in vivo measurement of pH. We expressed three different GEpHIs in the cytosol of Drosophila larval motor neurons and observed substantial presynaptic acidification in nerve termini during nerve stimulation in situ. SuperEcliptic pHluorin was the most useful GEpHI for studying pHcyto shifts in this model system. We determined the resting pH of the nerve terminal cytosol to be 7.30 ± 0.02, and observed a decrease of 0.16 ± 0.01 pH units when the axon was stimulated at 40 Hz for 4 s. Realkalinization occurred upon cessation of stimulation with a time course of 20.54 ± 1.05 s (τ). The chemical pH-indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein corroborated these changes in pHcyto. Bicarbonate-derived buffering did not contribute to buffering of acid loads from short (≤4 s) trains of action potentials but did buffer slow (∼60 s) acid loads. The magnitude of cytosolic acid transients correlated with cytosolic Ca2+ increase upon stimulation, and partial inhibition of the plasma membrane Ca2+-ATPase, a Ca2+/H+ exchanger, attenuated pHcyto shifts. Repeated stimulus trains mimicking motor patterns generated greater cytosolic acidification (∼0.30 pH units). Imaging through the cuticle of intact larvae revealed spontaneous pHcyto shifts in presynaptic termini in vivo, similar to those seen in situ during fictive locomotion, indicating that presynaptic pHcyto shifts cannot be dismissed as artifacts of ex vivo preparations. PMID:23401611

  10. Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90 (Hsp90): functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance.

    Science.gov (United States)

    Wang, Guan-Feng; Wei, Xuening; Fan, Renchun; Zhou, Huanbin; Wang, Xianping; Yu, Chunmei; Dong, Lingli; Dong, Zhenying; Wang, Xiaojie; Kang, Zhensheng; Ling, Hongqing; Shen, Qian-Hua; Wang, Daowen; Zhang, Xiangqi

    2011-07-01

    Heat shock protein 90 (Hsp90) molecular chaperones play important roles in plant growth and responses to environmental stimuli. However, little is known about the genes encoding Hsp90s in common wheat. Here, we report genetic and functional analysis of the genes specifying cytosolic Hsp90s in this species. Three groups of homoeologous genes (TaHsp90.1, TaHsp90.2 and TaHsp90.3), encoding three types of cytosolic Hsp90, were isolated. The loci containing TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes were assigned to groups 2, 7 and 5 chromosomes, respectively. TaHsp90.1 genes exhibited higher transcript levels in the stamen than in the leaf, root and culm. TaHsp90.2 and TaHsp90.3 genes were more ubiquitously transcribed in the vegetative and reproductive organs examined. Decreasing the expression of TaHsp90.1 genes through virus-induced gene silencing (VIGS) caused pronounced inhibition of wheat seedling growth, whereas the suppression of TaHsp90.2 or TaHsp90.3 genes via VIGS compromised the hypersensitive resistance response of the wheat variety Suwon 11 to stripe rust fungus. Our work represents the first systematic determination of wheat genes encoding cytosolic Hsp90s, and provides useful evidence for the functional involvement of cytosolic Hsp90s in the control of seedling growth and disease resistance in common wheat. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  11. A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA, but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival

    Directory of Open Access Journals (Sweden)

    Grischa Y. Chen

    2017-03-01

    Full Text Available Through unknown mechanisms, the host cytosol restricts bacterial colonization; therefore, only professional cytosolic pathogens are adapted to colonize this host environment. Listeria monocytogenes is a Gram-positive intracellular pathogen that is highly adapted to colonize the cytosol of both phagocytic and nonphagocytic cells. To identify L. monocytogenes determinants of cytosolic survival, we designed and executed a novel screen to isolate L. monocytogenes mutants with cytosolic survival defects. Multiple mutants identified in the screen were defective for synthesis of menaquinone (MK, an essential molecule in the electron transport chain. Analysis of an extensive set of MK biosynthesis and respiratory chain mutants revealed that cellular respiration was not required for cytosolic survival of L. monocytogenes but that, instead, synthesis of 1,4-dihydroxy-2-naphthoate (DHNA, an MK biosynthesis intermediate, was essential. Recent discoveries showed that modulation of the central metabolism of both host and pathogen can influence the outcome of host-pathogen interactions. Our results identify a potentially novel function of the MK biosynthetic intermediate DHNA and specifically highlight how L. monocytogenes metabolic adaptations promote cytosolic survival and evasion of host immunity.

  12. Unlocking Barriers to DNA Vaccine Immunogenicity: A Cross-Species Analysis of Cytosolic DNA Sensing in Skeletal Muscle Myocytes

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-15-1-0505 TITLE: Unlocking Barriers to DNA Vaccine Immunogenicity: A Cross-Species Analysis of Cytosolic DNA Sensing in...REPORT TYPE Annual 3. DATES COVERED 10 Sept 2016 – 9 Sept 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Unlocking Barriers to DNA Vaccine ...DISTRIBUTION / AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT DNA vaccine technology holds

  13. Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC-NLR proteins.

    Science.gov (United States)

    Cesari, Stella; Moore, John; Chen, Chunhong; Webb, Daryl; Periyannan, Sambasivam; Mago, Rohit; Bernoux, Maud; Lagudah, Evans S; Dodds, Peter N

    2016-09-06

    Plants possess intracellular immune receptors designated "nucleotide-binding domain and leucine-rich repeat" (NLR) proteins that translate pathogen-specific recognition into disease-resistance signaling. The wheat immune receptors Sr33 and Sr50 belong to the class of coiled-coil (CC) NLRs. They confer resistance against a broad spectrum of field isolates of Puccinia graminis f. sp. tritici, including the Ug99 lineage, and are homologs of the barley powdery mildew-resistance protein MLA10. Here, we show that, similarly to MLA10, the Sr33 and Sr50 CC domains are sufficient to induce cell death in Nicotiana benthamiana Autoactive CC domains and full-length Sr33 and Sr50 proteins self-associate in planta In contrast, truncated CC domains equivalent in size to an MLA10 fragment for which a crystal structure was previously determined fail to induce cell death and do not self-associate. Mutations in the truncated region also abolish self-association and cell-death signaling. Analysis of Sr33 and Sr50 CC domains fused to YFP and either nuclear localization or nuclear export signals in N benthamiana showed that cell-death induction occurs in the cytosol. In stable transgenic wheat plants, full-length Sr33 proteins targeted to the cytosol provided rust resistance, whereas nuclear-targeted Sr33 was not functional. These data are consistent with CC-mediated induction of both cell-death signaling and stem rust resistance in the cytosolic compartment, whereas previous research had suggested that MLA10-mediated cell-death and disease resistance signaling occur independently, in the cytosol and nucleus, respectively.

  14. Flexible change and cooperation between mitochondrial electron transport and cytosolic glycolysis as the basis for chilling tolerance in tomato plants.

    Science.gov (United States)

    Shi, Kai; Fu, Li-Jun; Zhang, Shuai; Li, Xin; Liao, Yang-Wen-Ke; Xia, Xiao-Jian; Zhou, Yan-Hong; Wang, Rong-Qing; Chen, Zhi-Xiang; Yu, Jing-Quan

    2013-02-01

    To find if cytosolic glycolysis dynamical metabolism plays a role in mediating respiration homeostasis and its relationship with mitochondrial electron transport chain (miETC) flexibility, we selected two tomato genotypes that differ in chilling tolerance and compared the responses of miETC, cytosolic glycolysis and respiratory homeostasis at 7 °C. Our results showed that the transcripts of both classical and bypass component genes for miETC and glycolysis were comparable for both genotypes when grown at 25 °C. However, there was a rapid global increase in the expression of most respiratory genes in response to chilling at 7 °C for both genotypes. When normally grown plant was set as the control for each genotype, the transcripts of most COX family members, ATP synthase, AOX1b, and UCP are highly up-regulated in chilling-tolerant Zhefen No. 208 plants in contrast to the sensitive Zhefen No. 212 plants. Both genotypes mobilized the energy-saving sucrose synthase pathway for sucrose degradation by cytosolic glycolysis, but this mechanism is evidently more effective in tolerant Zhefen No. 208 plants. Furthermore, only Zhefen No. 208 plants were able to partially switch from low-energy efficiency pathways to ATP conserving pathways to carry out fructose-6-phosphate conversion and pyruvate production. This metabolic flexibility in miETC and cytosolic glycolysis were coupled to higher ATP synthesis and lower ROS accumulation, which may be essential for sustaining the higher leaf respiration and homeostasis of chilling-tolerant plants.

  15. A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC)*

    Science.gov (United States)

    Schmidt, Axel; Löhrer, Daniel; Alsop, Richard J.; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Wirtz, Monika; Rheinstädter, Maikel C.; Gründer, Stefan; Wiemuth, Dominik

    2016-01-01

    The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na+ channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction. PMID:27679529

  16. Dictyostelium acetoacetyl-CoA thiolase is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol.

    Science.gov (United States)

    Isezaki, Nana; Sekiba, Atsushi; Itagaki, Shoko; Nagayama, Koki; Ochiai, Hiroshi; Ohmachi, Tetsuo

    2015-07-01

    Acetoacetyl-CoA thiolase is an enzyme that catalyses both the CoA-dependent thiolytic cleavage of acetoacetyl-CoA and the reverse condensation reaction. In Dictyostelium discoideum, acetoacetyl-CoA thiolase (DdAcat) is encoded by a single acat gene. The aim of this study was to assess the localization of DdAcat and to determine the mechanism of its cellular localization. Subcellular localization of DdAcat was investigated using a fusion protein with GFP, and it was found to be localized to peroxisomes. The findings showed that the targeting signal of DdAcat to peroxisomes is a unique nonapeptide sequence (15RMYTTAKNL23) similar to the conserved peroxisomal targeting signal-2 (PTS-2). Cell fractionation experiments revealed that DdAcat also exists in the cytosol. Distribution to the cytosol was caused by translational initiation from the second Met codon at position 16. The first 18 N-terminal residues also exhibited function as a mitochondrial targeting signal (MTS). These results indicate that DdAcat is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol using both PTS-2 and MTS signals, which overlap each other near the N-terminus, and the alternative utilization of start codons.

  17. WRKY62 transcription factor acts downstream of cytosolic NPR1 and negatively regulates jasmonate-responsive gene expression.

    Science.gov (United States)

    Mao, Peng; Duan, Mingrui; Wei, Chunhong; Li, Yi

    2007-06-01

    Cytosolic NPR1 has been shown to be essential for the salicylic acid (SA)-mediated suppression of jasmonic acid (JA)-responsive gene expression. However, factors downstream of NPR1 in the cross-talk between SA and JA signaling are unclear. Here we show that Arabidopsis WRKY62, a member of WRKY group III transcription factors, was induced by methyl jasmonate (MeJA) and SA treatment. The presence of basal SA is required for the MeJA-induced WRKY62 expression, and both chemicals exhibit a synergistic effect on WRKY62 induction. In addition, upon treatment with an extremely low concentration of SA, cytosolic NPR1 controls the MeJA-induced expression of WRKY62. TGA transcription factors, which up-regulate SA-induced expression of WRKY62, are dispensable for the induction of WRKY62 in JA signaling. Genetic dissection of both wrky62 mutants and WRKY62-overexpressing plants indicated that WRKY62 down-regulates JA-responsive LOX2 and VSP2 expression. Our results demonstrate that WRKY62 acts downstream of cytosolic NPR1 and negatively regulates JA-responsive gene expression, suggesting that WRKY62 may be involved in the SA-mediated suppression of JA signaling.

  18. Arf and RhoA regulate both the cytosolic and the membrane-bound phospholipase D from human placenta

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hansen, Harald S.; Provost, J.J.

    1997-01-01

    In this paper we demonstrate for the first time that human placenta contains a cytosolic phospholipase D (PLD) activity. This activity had a pH optimum of 7.0 and was stimulated by PIP and inhibited by oleate. Furthermore, cytosolic PLD was stimulated by 30 µM GTP¿S (6-14-fold) and by the small G...... proteins 1 µM mArf3 (2-fold) and 0.37 nM RhoA (2-fold). This is the first report to show RhoA activation of a cytosolic PLD. The activation by mArf3 was maintained after partial purification on DEAE Sepharose of the enzyme. We have previously reported the existence of a membrane-bound PLD from human...... placenta, which is stimulated by PIP, but not by oleate. Here we show that oleic acid and a-linolenic acid both dose-dependently inhibited solubilized membrane PLD (65% inhibition at 4 mM), whereas stearic acid (4 mM) had no effect. Thus, the presence of double bonds in the fatty acid is important...

  19. Tetanus toxin inhibits neurotensin-induced mobilization of cytosolic protein kinase C activity in NG-108 cells.

    Science.gov (United States)

    Considine, R V; Handler, C M; Simpson, L L; Sherwin, J R

    1991-01-01

    There is considerable literature on the pathogenesis of tetanus toxin poisoning; however, the mechanism of action and intracellular substrate of this toxin have not been defined. It was demonstrated that the NG-108 neuroblastoma x glioma cell line is a suitable model in which to study the mechanism of tetanus toxin action, from binding of the toxin to inhibition of transmitter release. Further, it has been shown that tetanus toxin pretreatment attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C (PKC) in this cell line. In the present study a 4-hr tetanus toxin pretreatment (10(-10)-10(-13) M) completely inhibited the mobilization of cytosolic PKC induced by a 30-min exposure to 10 microM neurotensin. Pretreatment with 10(-10) M tetanus toxin for periods as short as 1 hr was sufficient to attenuate the ability of neurotensin to mobilize cytosolic PKC; however, a 30-min pretreatment had no significant effect. At a concentration of 10(-11) M, it was necessary to pretreat the cells for greater than 1 hr to significantly attenuate neurotensin-mobilized PKC activity. The exact role that PKC plays in the secretory process is not yet known; however, these findings suggest that the effect of tetanus toxin on neurotransmitter release is accompanied by an alteration in PKC metabolism in differentiated NG-108 cells.

  20. Reduction of the cytosolic phosphoglucomutase in Arabidopsis reveals impact on plant growth, seed and root development, and carbohydrate partitioning.

    Directory of Open Access Journals (Sweden)

    Irina Malinova

    Full Text Available Phosphoglucomutase (PGM catalyses the interconversion of glucose 1-phosphate (G1P and glucose 6-phosphate (G6P and exists as plastidial (pPGM and cytosolic (cPGM isoforms. The plastidial isoform is essential for transitory starch synthesis in chloroplasts of leaves, whereas the cytosolic counterpart is essential for glucose phosphate partitioning and, therefore, for syntheses of sucrose and cell wall components. In Arabidopsis two cytosolic isoforms (PGM2 and PGM3 exist. Both PGM2 and PGM3 are redundant in function as single mutants reveal only small or no alterations compared to wild type with respect to plant primary metabolism. So far, there are no reports of Arabidopsis plants lacking the entire cPGM or total PGM activity, respectively. Therefore, amiRNA transgenic plants were generated and used for analyses of various parameters such as growth, development, and starch metabolism. The lack of the entire cPGM activity resulted in a strongly reduced growth revealed by decreased rosette fresh weight, shorter roots, and reduced seed production compared to wild type. By contrast content of starch, sucrose, maltose and cell wall components were significantly increased. The lack of both cPGM and pPGM activities in Arabidopsis resulted in dwarf growth, prematurely die off, and inability to develop a functional inflorescence. The combined results are discussed in comparison to potato, the only described mutant with lack of total PGM activity.

  1. Cytosolic Pellino-1-Mediated K63-Linked Ubiquitination of IRF5 in M1 Macrophages Regulates Glucose Intolerance in Obesity

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    Donghyun Kim

    2017-07-01

    Full Text Available IRF5 is a signature transcription factor that induces M1 macrophage polarization. However, little is known regarding cytosolic proteins that induce IRF5 activation for M1 polarization. Here, we report the interaction between ubiquitin E3 ligase Pellino-1 and IRF5 in the cytoplasm, which increased nuclear translocation of IRF5 by K63-linked ubiquitination in human and mouse M1 macrophages. LPS and/or IFN-γ increased Pellino-1 expression, and M1 polarization was attenuated in Pellino-1-deficient macrophages in vitro and in vivo. Defective M1 polarization in Pellino-1-deficient macrophages improved glucose intolerance in mice fed a high-fat diet. Furthermore, macrophages in adipose tissues from obese humans exhibited increased Pellino-1 expression and IRF5 nuclear translocation compared with nonobese subjects, and these changes are associated with insulin resistance index. This study demonstrates that cytosolic Pellino-1-mediated K63-linked ubiquitination of IRF5 in M1 macrophages regulates glucose intolerance in obesity, suggesting a cytosolic mediator function of Pellino-1 in TLR4/IFN-γ receptor-IRF5 axis during M1 polarization.

  2. Fluid shear stress modulates cytosolic free calcium in vascular endothelial cells.

    Science.gov (United States)

    Shen, J; Luscinskas, F W; Connolly, A; Dewey, C F; Gimbrone, M A

    1992-02-01

    Cytosolic free Ca2+ concentration ([Ca2+]i) was monitored in single and groups of fura-2-loaded bovine aortic endothelial cells (BAEC) during exposure to laminar fluid shear stress. Application of a step increase in shear stress from 0.08 to 8 dyn/cm2 to confluent BAEC monolayers resulted in a transient increase in [Ca2+]i, which attained a peak value in 15-40 s, followed by a decline to baseline within 40-80 s. The magnitude of the [Ca2+]i responses increased with applied shear stress over the range of 0.2-4 dyn/cm2 and reached a maximum at greater than 4 dyn/cm2. Transient oscillations in [Ca2+]i with gradually diminishing amplitude were observed in individual cells subjected to continuous high shear stress. Elimination of extracellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, blockade of Ca2+ entry with lanthanum, depolarization of the cell membrane with high K+, and preconditioning of BAEC in steady laminar flow had little effect on the [Ca2+]i response. In the presence of ATP or ADP, application of shear stress caused repetitive oscillations in [Ca2+]i in single BAEC, whose frequency was dependent on both agonist concentration and the magnitude of applied shear stress. However, apyrase, an ATPase and ADPase, did not inhibit the shear-induced [Ca2+]i responses in standard medium (no added ATP or ADP), suggesting that the shear-induced [Ca2+]i response is not due to ATP released by endothelial cells.

  3. The cytosolic glyoxalases of Plasmodium falciparum are dispensable during asexual blood-stage development

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    Cletus A. Wezena

    2017-11-01

    Full Text Available The enzymes glyoxalase 1 and 2 (Glo1 and Glo2 are found in most eukaryotes and catalyze the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Four glyoxalases are encoded in the genome of the malaria parasite Plasmodium falciparum, the cytosolic enzymes PfGlo1 and PfcGlo2, the apicoplast enzyme PftGlo2, and an inactive Glo1-like protein that also carries an apicoplast-targeting sequence. Inhibition or knockout of the Plasmodium glyoxalases was hypothesized to lead to an accumulation of 2-oxoaldehydes and advanced glycation end-products (AGE in the host-parasite unit and to result in parasite death. Here, we generated clonal P. falciparum strain 3D7 knockout lines for PFGLO1 and PFcGLO2 using the CRISPR-Cas9 system. Although 3D7Δglo1 knockout clones had an increased susceptibility to external glyoxal, all 3D7Δglo1 and 3D7Δcglo2 knockout lines were viable and showed no significant growth phenotype under standard growth conditions. Furthermore, the lack of PfcGlo2, but not PfGlo1, increased gametocyte commitment in the knockout lines. In summary, PfGlo1 and PfcGlo2 are dispensable during asexual blood-stage development while the loss of PfcGlo2 may induce the formation of transmissible gametocytes. These combined data show that PfGlo1 and PfcGlo2 are most likely not suited as targets for selective drug development.

  4. Cytosolic phospholipase A2α mediates Pseudomonas aeruginosa LPS-induced airway constriction of CFTR -/- mice

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    Lagranderie Micheline

    2010-04-01

    Full Text Available Abstract Background Lungs of cystic fibrosis (CF patients are chronically infected with Pseudomonas aeruginosa. Increased airway constriction has been reported in CF patients but underplaying mechanisms have not been elucidated. Aim: to examine the effect of P. aeruginosa LPS on airway constriction in CF mice and the implication in this process of cytosolic phospholipase A2α (cPLA2α, an enzyme involved in arachidonic acid (AA release. Methods Mice were instilled intra-nasally with LPS. Airway constriction was assessed using barometric plethysmograph. MIP-2, prostaglandin E2 (PGE2, leukotrienes and AA concentrations were measured in BALF using standard kits and gas chromatography. Results LPS induced enhanced airway constriction and AA release in BALF of CF compared to littermate mice. This was accompanied by increased levels of PGE2, but not those of leukotrienes. However, airway neutrophil influx and MIP-2 production remained similar in both mouse strains. The cPLA2α inhibitor arachidonyl trifluoro-methyl-ketone (ATK, but not aspirin which inhibit PGE2 synthesis, reduced LPS-induced airway constriction. LPS induced lower airway constriction and PGE2 production in cPLA2α -/- mice compared to corresponding littermates. Neither aspirin nor ATK interfered with LPS-induced airway neutrophil influx or MIP-2 production. Conclusions CF mice develop enhanced airway constriction through a cPLA2α-dependent mechanism. Airway inflammation is dissociated from airway constriction in this model. cPLA2α may represent a suitable target for therapeutic intervention in CF. Attenuation of airway constriction by cPLA2α inhibitors may help to ameliorate the clinical status of CF patients.

  5. NMR structure of integrin α4 cytosolic tail and its interactions with paxillin.

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    Geok-Lin Chua

    Full Text Available BACKGROUND: Integrins are a group of transmembrane signaling proteins that are important in biological processes such as cell adhesion, proliferation and migration. Integrins are α/β hetero-dimers and there are 24 different integrins formed by specific combinations of 18 α and 8 β subunits in humans. Generally, each of these subunits has a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT. CTs of integrins are important in bidirectional signal transduction and they associate with a large number of intracellular proteins. PRINCIPAL FINDINGS: Using NMR spectroscopy, we determined the 3-D structure of the full-length α4 CT (Lys968-Asp999 and characterize its interactions with the adaptor protein paxillin. The α4 CT assumes an overall helical structure with a kink in its membrane proximal region. Residues Gln981-Asn997 formed a continuous helical conformation that may be sustained by potential ionic and/or hydrogen bond interactions and packing of aromatic-aliphatic side-chains. ¹⁵N-¹H HSQC NMR experiments reveal interactions of the α4 CT C-terminal region with a fragment of paxillin (residues G139-K277 that encompassed LD2-LD4 repeats. Residues of these LD repeats including their adjoining linkers showed α4 CT binding-induced chemical shift changes. Furthermore, NMR studies using LD-containing peptides showed predominant interactions between LD3 and LD4 of paxillin and α4 CT. Docked structures of the α4 CT with these LD repeats suggest possible polar and/or salt-bridge and non-polar packing interactions. SIGNIFICANCE: The current study provides molecular insights into the structural diversity of α CTs of integrins and interactions of integrin α4 CT with the adaptor protein paxillin.

  6. Cytosolic proteome profiling of aminoglycosides resistant Mycobacterium tuberculosis clinical isolates using MALDI-TOF/MS

    Directory of Open Access Journals (Sweden)

    Divakar Sharma

    2016-11-01

    Full Text Available Emergence of extremely drug resistant tuberculosis (XDR-TB is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB. Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK and kanamycin (KM resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636 and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can

  7. Extracellular but not cytosolic superoxide dismutase protects against oxidant-mediated endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Erin L. Foresman

    2013-01-01

    Full Text Available Superoxide (O2•− contributes to the development of cardiovascular disease. Generation of O2•− occurs in both the intracellular and extracellular compartments. We hypothesized that the gene transfer of cytosolic superoxide dismutase (SOD1 or extracellular SOD (SOD3 to blood vessels would differentially protect against O2•−-mediated endothelial-dependent dysfunction. Aortic ring segments from New Zealand rabbits were incubated with adenovirus (Ad containing the gene for Escherichia coli β-galactosidase, SOD1, or SOD3. Activity assays confirmed functional overexpression of both SOD3 and SOD1 isoforms in aorta 24 h following gene transfer. Histochemical staining for β-galactosidase showed gene transfer occurred in the endothelium and adventitia. Next, vessels were prepared for measurement of isometric tension in Kreb's buffer containing xanthine. After precontraction with phenylephrine, xanthine oxidase impaired relaxation to the endothelium-dependent dilator acetylcholine (ACh, max relaxation 33±4% with XO vs. 64±3% without XO, p<0.05, whereas relaxation to the endothelium-independent dilator sodium nitroprusside was unaffected. In the presence of XO, maximal relaxation to ACh was improved in vessels incubated with AdSOD3 (55±2%, p<0.05 vs. control but not AdSOD1 (34±4%. We conclude that adenoviral-mediated gene transfer of SOD3, but not SOD1, protects the aorta from xanthine/XO-mediated endothelial dysfunction. These data provide important insight into the location and enzymatic source of O2•− production in vascular disease.

  8. Cytosolic streaming in vegetative mycelium and aerial structures of Aspergillus niger.

    Science.gov (United States)

    Bleichrodt, R; Vinck, A; Krijgsheld, P; van Leeuwen, M R; Dijksterhuis, J; Wösten, H A B

    2013-03-15

    Aspergillus niger forms aerial hyphae and conidiophores after a period of vegetative growth. The hyphae within the mycelium of A. niger are divided by septa. The central pore in these septa allows for cytoplasmic streaming. Here, we studied inter- and intra-compartmental streaming of the reporter protein GFP in A. niger. Expression of the gene encoding nuclear targeted GFP from the gpdA or glaA promoter resulted in strong fluorescence of nuclei within the vegetative hyphae and weak fluorescence in nuclei within the aerial structures. These data and nuclear run on experiments showed that gpdA and glaA are higher expressed in the vegetative mycelium when compared to aerial hyphae, conidiophores and conidia. Notably, gpdA or glaA driven expression of the gene encoding cytosolic GFP resulted in strongly fluorescent vegetative hyphae and aerial structures. Apparently, GFP streams from vegetative hyphae into aerial structures. This was confirmed by monitoring fluorescence of photo-activatable GFP (PA-GFP). In contrast, PA-GFP did not stream from aerial structures to vegetative hyphae. Streaming of PA-GFP within vegetative hyphae or within aerial structures of A. niger occurred at a rate of 10-15 μm s(-1). Taken together, these results not only show that GFP streams from the vegetative mycelium to aerial structures but it also indicates that its encoding RNA is not streaming. Absence of RNA streaming would explain why distinct RNA profiles were found in aerial structures and the vegetative mycelium by nuclear run on analysis and micro-array analysis.

  9. Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus

    Science.gov (United States)

    Johnson, R. F.; Beltz, T. G.; Sharma, R. V.; Xu, Z.; Bhatty, R. A.; Johnson, A. K.

    2001-01-01

    The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.

  10. Molecular insight into substrate recognition by human cytosolic sialidase NEU2.

    Science.gov (United States)

    Mozzi, Alessandra; Mazzacuva, Pietro; Zampella, Giuseppe; Forcella, Matilde Emma; Fusi, Paola Alessandra; Monti, Eugenio

    2012-04-01

    Sialidases or neuramidases are glycoside hydrolases removing terminal sialic acid residues from sialo-glycoproteins and sialo-glycolipids. Viral neuraminidases (NAs) have been extensively characterized and represent an excellent target for antiviral therapy through the synthesis of a series of competitive inhibitors that block the release of newly formed viral particles from infected cells. The human cytosolic sialidase NEU2 is the only mammalian enzyme structurally characterized and represents a valuable model to study the specificity of novel NA inhibitory drugs. Moreover, the availability of NEU2 3D structure represents a pivotal step toward the characterization of the molecular basis of natural substrates recognition by the enzyme. In this perspective, we have carried out a study of molecular docking of NEU2 active site using natural substrates of increasing complexity. Moreover, selective mutations of the residues putatively involved into substrate(s) interaction/recognition have been performed, and the resulting mutant enzymes have been preliminary tested for their catalytic activity and substrate specificity. We found that Q270 is involved in the binding of the disaccharide α(2,3) sialyl-galactose, whereas K45 and Q112 bind the distal glucose of the trisaccharide α(2,3) sialyl-lactose, corresponding to the oligosaccharide moiety of GM3 ganglioside. In addition, E218, beside D46, is proved to be a key catalytic residue, being, together with Y334, the second member of the nucleophile pair required for the catalysis. Overall, our results point out the existence of a dynamic network of interactions that are possibly involved in the recognition of the glycans bearing sialic acid. Copyright © 2011 Wiley Periodicals, Inc.

  11. LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

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    Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany); Brosens, Jan [Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry CV2 2DX (United Kingdom); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany)

    2015-05-08

    Objective: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H{sup +} and lactate. H{sup +} extrusion is in part accomplished by Na{sup +}/H{sup +} exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. Methods: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pH{sub i} estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na{sup +}/H{sup +} exchanger activity from Na{sup +} dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. Results: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na{sup +}/H{sup +} exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). Conclusions: LeftyA markedly down-regulates NHE1 expression, Na{sup +}/H{sup +} exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition. - Highlights: • LeftyA, an inhibitor of tumor growth, reduces Na{sup +}/H{sup +}-exchanger activity by 55%. • LeftyA decreases NHE1 transcripts by 99.6% and NHE1 protein by 71%. • LeftyA decreases cytosolic pH from 7.22 ± 0.02 to 7.05 ± 0.02. • Cytosolic acidification by Lefty A decreases glycolysis by 41%. • Cytosolic acidification by Lefty A compromises energy production of tumor cells.

  12. [Experimental studies on seborrheic alopecia. II. Determination of the structural differences between testosterone transported cytosol protein of the bald and hairy areas].

    Science.gov (United States)

    Bassas, E

    1975-01-01

    By previous isolation of the testosterone transport cytosol protein in bald and hairy areas in patients with male pattern baldness, the author studies the differences concerning the protein citosol-testosterone complex, after fixation by means of labelled testosterone and electrophoresis, measuring the corresponding radioactivity curves. The results show that there are important structural differences for the cytosol protein between the hairy and bald regions.

  13. Protein kinase C modulates cytosolic free calcium by stimulating calcium pump activity in Jurkat T cells.

    Science.gov (United States)

    Balasubramanyam, M; Gardner, J P

    1995-12-01

    Although protein kinase C (PKC) activation has been shown to inhibit Ca2+ influx in T lymphocytes, the role of PKC on Ca2+ sequestration or extrusion processes has not been fully explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca2+ (Ca2+i) extrusion and 45Ca2+ efflux in human leukemic Jurkat T cells. Treatment of Fura-2 loaded cells with phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (THYM) resulted in a decrease in Ca2+i both in the presence and absence of extracellular Ca2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca2+i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca2+i to basal levels by 17-53%. PKC stimulation of the Ca2+i decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca2+i decline in TG-treated cells, antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca2+i extrusion were further explored. PMA significantly increased the rate of Ca2+ extrusion in TG-treated cells from 0.28 +/- 0.02 to 0.35 +/- 0.03 s-1 (mean +/- SEM) and stimulated the initial rate of 45Ca2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca2+i decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca2+ pump. In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response. We suggest that Jurkat T cells contain a PKC-sensitive Ca2+ extrusion mechanism likely to be the Ca2+ pump. In lymphocytes, receptor/PLC-linked PKC activation modulates Ca2+i not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca2+i

  14. A Comparison of Two Yeast MnSODs: Mitochondrial Saccharomyces cerevisiae versus Cytosolic Candida albicans

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    Sheng Y.; Cabelli D.; Stich, T.A.; Barnese, K.; Gralla, E.B.; Cascio, D.; Britt, R.D.; Valentine, J.S.

    2011-12-28

    Human MnSOD is significantly more product-inhibited than bacterial MnSODs at high concentrations of superoxide (O{sub 2}{sup -}). This behavior limits the amount of H{sub 2}O{sub 2} produced at high [O{sub 2}{sup -}]; its desirability can be explained by the multiple roles of H{sub 2}O{sub 2} in mammalian cells, particularly its role in signaling. To investigate the mechanism of product inhibition in MnSOD, two yeast MnSODs, one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), were isolated and characterized. ScMnSOD and CaMnSODc are similar in catalytic kinetics, spectroscopy, and redox chemistry, and they both rest predominantly in the reduced state (unlike most other MnSODs). At high [O{sub 2}{sup -}], the dismutation efficiencies of the yeast MnSODs surpass those of human and bacterial MnSODs, due to very low level of product inhibition. Optical and parallel-mode electron paramagnetic resonance (EPR) spectra suggest the presence of two Mn{sup 3+} species in yeast Mn{sup 3+}SODs, including the well-characterized 5-coordinate Mn{sup 3+} species and a 6-coordinate L-Mn{sup 3+} species with hydroxide as the putative sixth ligand (L). The first and second coordination spheres of ScMnSOD are more similar to bacterial than to human MnSOD. Gln154, an H-bond donor to the Mn-coordinated solvent molecule, is slightly further away from Mn in yeast MnSODs, which may result in their unusual resting state. Mechanistically, the high efficiency of yeast MnSODs could be ascribed to putative translocation of an outer-sphere solvent molecule, which could destabilize the inhibited complex and enhance proton transfer from protein to peroxide. Our studies on yeast MnSODs indicate the unique nature of human MnSOD in that it predominantly undergoes the inhibited pathway at high [O{sub 2}{sup -}].

  15. Trolox-sensitive reactive oxygen species regulate mitochondrial morphology, oxidative phosphorylation and cytosolic calcium handling in healthy cells.

    Science.gov (United States)

    Distelmaier, Felix; Valsecchi, Federica; Forkink, Marleen; van Emst-de Vries, Sjenet; Swarts, Herman G; Rodenburg, Richard J T; Verwiel, Eugène T P; Smeitink, Jan A M; Willems, Peter H G M; Koopman, Werner J H

    2012-12-15

    Cell regulation by signaling reactive oxygen species (sROS) is often incorrectly studied through extracellular oxidant addition. Here, we used the membrane-permeable antioxidant Trolox to examine the role of sROS in mitochondrial morphology, oxidative phosphorylation (OXPHOS), and cytosolic calcium (Ca(2+)) handling in healthy human skin fibroblasts. Trolox treatment reduced the levels of 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein (CM-H(2)DCF) oxidizing ROS, lowered cellular lipid peroxidation, and induced a less oxidized mitochondrial thiol redox state. This was paralleled by increased glutathione- and mitofusin-dependent mitochondrial filamentation, increased expression of fully assembled mitochondrial complex I, elevated activity of citrate synthase and OXPHOS enzymes, and a higher cellular O(2) consumption. In contrast, Trolox did not alter hydroethidium oxidation, cytosolic thiol redox state, mitochondrial NAD(P)H levels, or mitochondrial membrane potential. Whole genome expression profiling revealed that Trolox did not trigger significant changes in gene expression, suggesting that Trolox acts downstream of this process. Cytosolic Ca(2+) transients, induced by the hormone bradykinin, were of a higher amplitude and decayed faster in Trolox-treated cells. These effects were dose-dependently antagonized by hydrogen peroxide. Our findings suggest that Trolox-sensitive sROS are upstream regulators of mitochondrial mitofusin levels, morphology, and function in healthy human skin fibroblasts. This information not only facilitates the interpretation of antioxidant effects in cell models (of oxidative-stress), but also contributes to a better understanding of ROS-related human pathologies, including mitochondrial disorders.

  16. A Cytosolic Acyltransferase Contributes to Triacylglycerol Synthesis in Sucrose-Rescued Arabidopsis Seed Oil Catabolism Mutants1[W][OA

    Science.gov (United States)

    Hernández, M. Luisa; Whitehead, Lynne; He, Zhesi; Gazda, Valeria; Gilday, Alison; Kozhevnikova, Ekaterina; Vaistij, Fabián E.; Larson, Tony R.; Graham, Ian A.

    2012-01-01

    Triacylglycerol (TAG) levels and oil bodies persist in sucrose (Suc)-rescued Arabidopsis (Arabidopsis thaliana) seedlings disrupted in seed oil catabolism. This study set out to establish if TAG levels persist as a metabolically inert pool when downstream catabolism is disrupted, or if other mechanisms, such as fatty acid (FA) recycling into TAG are operating. We show that TAG composition changes significantly in Suc-rescued seedlings compared with that found in dry seeds, with 18:2 and 18:3 accumulating. However, 20:1 FA is not efficiently recycled back into TAG in young seedlings, instead partitioning into the membrane lipid fraction and diacylglycerol. In the lipolysis mutant sugar dependent1and the β-oxidation double mutant acx1acx2 (for acyl-Coenzyme A oxidase), levels of TAG actually increased in seedlings growing on Suc. We performed a transcriptomic study and identified up-regulation of an acyltransferase gene, DIACYLGLYCEROL ACYLTRANSFERASE3 (DGAT3), with homology to a peanut (Arachis hypogaea) cytosolic acyltransferase. The acyl-Coenzyme A substrate for this acyltransferase accumulates in mutants that are blocked in oil breakdown postlipolysis. Transient expression in Nicotiana benthamiana confirmed involvement in TAG synthesis and specificity toward 18:3 and 18:2 FAs. Double-mutant analysis with the peroxisomal ATP-binding cassette transporter mutant peroxisomal ABC transporter1 indicated involvement of DGAT3 in the partitioning of 18:3 into TAG in mutant seedlings growing on Suc. Fusion of the DGAT3 protein with green fluorescent protein confirmed localization to the cytosol of N. benthamiana. This work has demonstrated active recycling of 18:2 and 18:3 FAs into TAG when seed oil breakdown is blocked in a process involving a soluble cytosolic acyltransferase. PMID:22760209

  17. Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding

    Science.gov (United States)

    Shi, Yunhua; Mowery, Richard A; Ashley, Jonathan; Hentz, Michelle; Ramirez, Alejandro J; Bilgicer, Basar; Slunt-Brown, Hilda; Borchelt, David R; Shaw, Bryan F

    2012-01-01

    The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of “gel shifting” has perplexed biochemists and biologists for decades. We identify a mechanism for “gel shifting” that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues ∼80–101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80–101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80–101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic). PMID:22692797

  18. Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response.

    Directory of Open Access Journals (Sweden)

    Sébastien Plumet

    Full Text Available BACKGROUND: Double stranded RNA (dsRNA is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA. METHODOLOGY AND PRINCIPAL FINDINGS: During measles virus infection, the IFN-beta gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-beta gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-beta expression, provided that it was delivered into the cytosol as a 5'-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-beta induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5'-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5'-triphosphate RNA end. CONCLUSIONS/SIGNIFICANCE: RIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5'-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader.

  19. Bmal1 is an essential regulator for circadian cytosolic Ca²⁺ rhythms in suprachiasmatic nucleus neurons.

    Science.gov (United States)

    Ikeda, Masayuki; Ikeda, Masaaki

    2014-09-03

    The hypothalamic suprachiasmatic nucleus (SCN) plays a pivotal role in the mammalian circadian clock system. Bmal1 is a clock gene that drives transcriptional-translational feedback loops (TTFLs) for itself and other genes, and is expressed in nearly all SCN neurons. Despite strong evidence that Bmal1-null mutant mice display arrhythmic behavior under constant darkness, the function of Bmal1 in neuronal activity is unknown. Recently, periodic changes in the levels of intracellular signaling messengers, such as cytosolic Ca(2+) and cAMP, were suggested to regulate TTFLs. However, the opposite aspect of how clock gene TTFLs regulate cytosolic signaling remains unclear. To investigate intracellular Ca(2+) dynamics under Bmal1 perturbations, we cotransfected some SCN neurons with yellow cameleon together with wild-type or dominant-negative Bmal1 using a gene-gun applied for mouse organotypic cultures. Immunofluorescence staining for a tag protein linked to BMAL1 showed nuclear expression of wild-type BMAL1 and its degradation within 1 week after transfection in SCN neurons. However, dominant-negative BMAL1 did not translocate into the nucleus and the cytosolic signals persisted beyond 1 week. Consistently, circadian Ca(2+) rhythms in SCN neurons were inhibited for longer periods by dominant-negative Bmal1 overexpression. Furthermore, SCN neurons transfected with a Bmal1 shRNA lengthened, whereas those overexpressing wild-type Bmal1 shortened, the periods of Ca(2+) rhythms, with a significant reduction in their amplitude. BMAL1 expression was intact in the majority of neighboring neurons in organotypic cultures. Therefore, we conclude that proper intrinsic Bmal1 expression, but not passive signaling via cell-to-cell interactions, is the determinant of circadian Ca(2+) rhythms in SCN neurons. Copyright © 2014 the authors 0270-6474/14/3412029-10$15.00/0.

  20. The deposition of suberin lamellae determines the magnitude of cytosolic Ca2+ elevations in root endodermal cells subjected to cooling.

    Science.gov (United States)

    Moore, Catherine A; Bowen, Helen C; Scrase-Field, Sarah; Knight, Marc R; White, Philip J

    2002-05-01

    A transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt) is thought to be a prerequisite for an appropriate physiological response to both chilling and salt stress. The [Ca2+]cyt is raised by Ca2+ influx to the cytosol from the apoplast and/or intracellular stores. It has been speculated that different signals mobilise Ca2+ from different stores, but little is known about the origin(s) of the Ca2+ entering the cytosol in response to specific environmental challenges. We have utilised the developmentally regulated suberisation of endodermal cells, which is thought to prevent Ca2+ influx from the apoplast, to ascertain whether Ca2+ influx is required to increase [Ca2+]cyt in response to chilling or salt stress. Perturbations in [Ca2+]cyt were studied in transgenic Arabidopsis thaliana, expressing aequorin fused to a modified yellow fluorescent protein solely in root endodermal cells, during slow cooling of plants from 20 to 0.5 degrees C over 5 min and in response to an acute salt stress (0.333 m NaCl). Only in endodermal cells in the apical 4 mm of the Arabidopsis root did [Ca2+]cyt increase significantly during cooling, and the magnitude of the [Ca2+]cyt elevation elicited by cooling was inversely related to the extent of suberisation of the endodermal cell layer. No [Ca2+]cyt elevations were elicited by cooling in suberised endodermal cells. This is consistent with the hypothesis that suberin lamellae isolate the endodermal cell protoplast from the apoplast and, thereby, prevent Ca2+ influx. By contrast, acute salt stress increased [Ca2+]cyt in endodermal cells throughout the root. These results suggest that [Ca2+]cyt elevations, upon slow cooling, depend absolutely on Ca2+ influx across the plasma membrane, but [Ca2+]cyt elevations in response to acute salt stress do not. They also suggest that Ca2+ release from intracellular stores contributes significantly to increasing [Ca2+]cyt upon acute salt stress.

  1. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Minh Vu Chuong, E-mail: mvchuong@yahoo.fr [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Zhang, Leilei [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Lhomme, Stanislas; Mouz, Nicolas [PX' Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble (France); Lenormand, Jean-Luc [HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Universite Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche (France); Lardy, Bernard; Morel, Francoise [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  2. Catalysis of nitro-aci tautomerism of the genotoxicant 2-nitropropane by cytosol from rodent and human liver.

    Science.gov (United States)

    Kohl, C; Gescher, A

    1996-01-05

    2-Nitropropane (2-NP) is a genotoxicant and hepatocarcinogen in rodents. Conversion to propane 2-nitronate (P2N), the anion of the tautomeric aci form of 2-NP, seems to be a pivotal part of the mechanism by which 2-NP causes its toxicity. We tested the hypothesis that the tautomeric equilibrium is influenced by enzymes in the liver, the target organ of 2-NP toxicity. Rat or mouse hepatocytes were incubated with 2-NP, P2N or the 2-NP isotopomer 2-deutero 2-nitropropane (2H-2-NP), which equilibrates with P2N much more slowly than 2-NP. Tautomers were analyzed by HPLC. The rates of conversion of 2-NP to P2N expressed as nmol P2N x (10(6) cells/ml)-1 x min-1 were 4.0 and 4.2 in the presence of hepatocytes from rats or mice, respectively, and 2.6 in the absence of cells. Production of 2-NP to P2N expressed as nmol 2-NP x (10(6) cells/ml)-1 x min-1 was increased from 6.1 in the absence of cells to 11.9 or 9.9 in the presence of hepatocytes from rats or mice, respectively. The rate of formation of P2N from 2H-2-NP as compared to 2-NP was characterised by a primary isotope effect of 3.4 and 3.8 in hepatocytes from rats and mice, respectively, contrasting with a value of 9.6 measured in medium omitting cells. When 2-NP was incubated with subfractions of rodent or human liver homogenate, production of P2N by cytosol was between 7.3 (mouse liver) and 28.1 times (human liver) higher than that observed in microsomes. Similarly generation of 2-NP from P2N by cytosol exceeded that in microsomes by a factor of two. Tautomerism in heat-activated cytosol, mitochondria or microsomes was not different from that in buffer only. The results suggest that the nitro-aci tautomerism of secondary nitroalkanes is catalysed by a hepatic enzyme which resides predominantly in the cytosol and may thus contribute to the generation of the toxic species via which 2-NP exerts its toxicity.

  3. Cytosolic entry of Shiga-like toxin a chain from the yeast endoplasmic reticulum requires catalytically active Hrd1p.

    Directory of Open Access Journals (Sweden)

    Shuyu Li

    Full Text Available BACKGROUND: Escherichia coli Shiga-like toxin 1 normally traffics to the endoplasmic reticulum (ER in sensitive mammalian cells from where the catalytic A chain (SLTxA1 dislocates to the cytosol to inactivate ribosomes. Currently, no molecular details of the dislocation process are available. To investigate the mechanism of the dislocation step we expressed SLTxA1 in the ER of Saccharomyces cerevisiae. METHODOLOGY AND PRINCIPAL FINDINGS: Using a combination of growth studies and biochemical tracking in yeast knock-out strains we show that SLTxA1 follows an ER-associated degradation (ERAD pathway to enter the cytosol in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex. ER-to-cytosol dislocation of the bulk population of SLTxA1 requires Cdc48p and its ubiquitin-handling co-factor Npl4p, and this population of toxin is terminally dispatched by proteasomal degradation. A small sub-population of SLTxA1 uncouples from this classical ERAD pathway and recovers catalytic activity in the cytosol. The pathway that leads to toxicity is also Hrd1p-dependent but, unlike that for the related ricin A chain toxin, SLTxA1 dislocation does require the catalytic cysteine of Hrd1p. However it does not depend on canonical ubiquitylation since toxin variants lacking endogenous lysyl residues also utilize this pathway, and furthermore there is no requirement for a number of Cdc48p co-factors. CONCLUSIONS AND SIGNIFICANCE: The fraction of SLTxA1 that disengages from the ERAD pathway thus does so upstream of Cdc48p interactions and downstream of Hrd1p interactions, in a step that possibly involves de-ubiquitylation. Mechanistically therefore, the dislocation of this toxin is quite distinct from that of conventional ERAD substrates that are normally degraded, and the toxins partially characterised to date that do not require the catalytic cysteine of the major Hrd1p component of the dislocation apparatus.

  4. Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Bosche, Bert, E-mail: bert.bosche@uk-essen.de [Department of Neurology, University of Duisburg-Essen (Germany); Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne (Germany); Schäfer, Matthias, E-mail: matthias.schaefer@sanofi.com [Institute of Physiology, Justus-Liebig-University Giessen (Germany); Graf, Rudolf, E-mail: rudolf.graf@nf.mpg.de [Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne (Germany); Härtel, Frauke V., E-mail: frauke.haertel@tu-dresden.de [Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden (Germany); Schäfer, Ute, E-mail: ute.schaefer@medunigraz.at [Research Unit for Experimental Neurotraumatology, Medical University of Graz (Austria); Noll, Thomas, E-mail: thomas.noll@tu-dresden.de [Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden (Germany)

    2013-05-03

    Highlights: •We investigate free calcium as a central signalling element in endothelial cells. •Inhibition of glycolysis with 2-deoxy-D-glucose reduces cellular ATP. •This manoeuvre leads to a biphasic increase and overload of free calcium. •Pre-treatment with lithium for 24 h abolishes both phases of the calcium increase. •This provides a new strategy to protect endothelial calcium homeostasis and barrier function. -- Abstract: Cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca{sup 2+}]{sub i} overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca{sup 2+}]{sub i} overload can be prevented by lithium treatment. [Ca{sup 2+}]{sub i} and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-D-glucose (5 mM; 2-DG) plus sodium cyanide (5 mM; NaCN) caused a significant decrease in cellular ATP content (14 ± 1 nmol/mg protein vs. 18 ± 1 nmol/mg protein in the control, n = 6 culture dishes, P < 0.05), an increase in [Ca{sup 2+}]{sub i} (278 ± 24 nM vs. 71 ± 2 nM in the control, n = 60 cells, P < 0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10 mM 2-DG led to a similar decrease in ATP content (14 ± 2 nmol/mg vs. 18 ± 1 nmol/mg in the control, P < 0.05) with a delay of 5 min. The [Ca{sup 2+}]{sub i} response of EC was biphasic with a peak after 1 min (183 ± 6 nM vs. 71 ± 1 nM, n = 60 cells, P < 0.05) followed by a sustained increase in [Ca{sup 2+}]{sub i}. A 24-h pre-treatment with 10 mM of lithium

  5. Sulfation of thyroid hormone by estrogen sulfotransferase

    NARCIS (Netherlands)

    M.H.A. Kester (Monique); T.J. Visser (Theo); C.H. van Dijk (Caren); D. Tibboel (Dick); A.M. Hood (Margaret); N.J. Rose; W. Meinl; U. Pabel; H. Glatt; C.N. Falany; M.W. Coughtrie

    1999-01-01

    textabstractSulfation is one of the pathways by which thyroid hormone is inactivated. Iodothyronine sulfate concentrations are very high in human fetal blood and amniotic fluid, suggesting important production of these conjugates in utero. Human estrogen

  6. Recombinant BCG Expressing ESX-1 of Mycobacterium marinum Combines Low Virulence with Cytosolic Immune Signaling and Improved TB Protection.

    Science.gov (United States)

    Gröschel, Matthias I; Sayes, Fadel; Shin, Sung Jae; Frigui, Wafa; Pawlik, Alexandre; Orgeur, Mickael; Canetti, Robin; Honoré, Nadine; Simeone, Roxane; van der Werf, Tjip S; Bitter, Wilbert; Cho, Sang-Nae; Majlessi, Laleh; Brosch, Roland

    2017-03-14

    Recent insights into the mechanisms by which Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is recognized by cytosolic nucleotide sensors have opened new avenues for rational vaccine design. The only licensed anti-tuberculosis vaccine, Mycobacterium bovis BCG, provides limited protection. A feature of BCG is the partial deletion of the ESX-1 type VII secretion system, which governs phagosomal rupture and cytosolic pattern recognition, key intracellular phenotypes linked to increased immune signaling. Here, by heterologously expressing the esx-1 region of Mycobacterium marinum in BCG, we engineered a low-virulence, ESX-1-proficient, recombinant BCG (BCG::ESX-1 Mmar ) that induces the cGas/STING/TBK1/IRF-3/type I interferon axis and enhances AIM2 and NLRP3 inflammasome activity, resulting in both higher proportions of CD8 + T cell effectors against mycobacterial antigens shared with BCG and polyfunctional CD4 + Th1 cells specific to ESX-1 antigens. Importantly, independent mouse vaccination models show that BCG::ESX-1 Mmar confers superior protection relative to parental BCG against challenges with highly virulent M. tuberculosis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Flightless-I, a gelsolin family member and transcriptional regulator, preferentially binds directly to activated cytosolic CaMK-II.

    Science.gov (United States)

    Seward, Matthew E; Easley, Charles A; McLeod, Jamie J; Myers, Alexandra L; Tombes, Robert M

    2008-07-23

    In order to evaluate links between Ca2+/calmodulin (CaM)-dependent protein kinase type II (CaMK-II) and cell cycle progression, CaMK-II binding partners were sought in proliferating cells by epitope-tag tandem mass spectrometry. One protein identified was the gelsolin family member, flightless-I (Fli-I). Fli-I is not a CaMK-II substrate, but binds directly and preferentially to constitutively active (T287D) CaMK-II over inactive CaMK-II. Fli-I gradually enters the nucleus upon CaMK-II inhibition and is retained in the cytosol by T287D CaMK-II. CaMK-II inhibition and Fli-I overexpression suppress transcription of beta-catenin dependent transcriptional reporters, whereas Fli-I suppression enhances their transcription. These findings support a novel mechanism whereby cytosolic CaMK-II influences beta-catenin dependent gene expression through Fli-I.

  8. Epi-reevesioside F inhibits Na+/K+-ATPase, causing cytosolic acidification, Bak activation and apoptosis in glioblastoma.

    Science.gov (United States)

    Hsu, Jui-Ling; Liu, Fan-Lun; Hsu, Lih-Ching; Chang, Hsun-Shuo; Leu, Wohn-Jenn; Yu, Chia-Chun; Chang, Wei-Ling; Chen, Ih-Sheng; Kung, Fan-Lu; Guh, Jih-Hwa

    2015-09-15

    Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC50 values of 27.3±1.7 vs. 48.7±1.8 nM (P Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na+/K+-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na+ but not Ca2+, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na+/K+-ATPase, leading to overload of intracellular Na+ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.

  9. Glutaredoxin 5 deficiency causes sideroblastic anemia by specifically impairing heme biosynthesis and depleting cytosolic iron in human erythroblasts.

    Science.gov (United States)

    Ye, Hong; Jeong, Suh Young; Ghosh, Manik C; Kovtunovych, Gennadiy; Silvestri, Laura; Ortillo, Danilo; Uchida, Naoya; Tisdale, John; Camaschella, Clara; Rouault, Tracey A

    2010-05-01

    Glutaredoxin 5 (GLRX5) deficiency has previously been identified as a cause of anemia in a zebrafish model and of sideroblastic anemia in a human patient. Here we report that GLRX5 is essential for iron-sulfur cluster biosynthesis and the maintenance of normal mitochondrial and cytosolic iron homeostasis in human cells. GLRX5, a mitochondrial protein that is highly expressed in erythroid cells, can homodimerize and assemble [2Fe-2S] in vitro. In GLRX5-deficient cells, [Fe-S] cluster biosynthesis was impaired, the iron-responsive element-binding (IRE-binding) activity of iron regulatory protein 1 (IRP1) was activated, and increased IRP2 levels, indicative of relative cytosolic iron depletion, were observed together with mitochondrial iron overload. Rescue of patient fibroblasts with the WT GLRX5 gene by transfection or viral transduction reversed a slow growth phenotype, reversed the mitochondrial iron overload, and increased aconitase activity. Decreased aminolevulinate delta, synthase 2 (ALAS2) levels attributable to IRP-mediated translational repression were observed in erythroid cells in which GLRX5 expression had been downregulated using siRNA along with marked reduction in ferrochelatase levels and increased ferroportin expression. Erythroblasts express both IRP-repressible ALAS2 and non-IRP-repressible ferroportin 1b. The unique combination of IRP targets likely accounts for the tissue-specific phenotype of human GLRX5 deficiency.

  10. Identification of host cytosolic sensors and bacterial factors regulating the type I interferon response to Legionella pneumophila.

    Directory of Open Access Journals (Sweden)

    Kathryn M Monroe

    2009-11-01

    Full Text Available Legionella pneumophila is a gram-negative bacterial pathogen that replicates in host macrophages and causes a severe pneumonia called Legionnaires' Disease. The innate immune response to L. pneumophila remains poorly understood. Here we focused on identifying host and bacterial factors involved in the production of type I interferons (IFN in response to L. pneumophila. It was previously suggested that the delivery of L. pneumophila DNA to the host cell cytosol is the primary signal that induces the type I IFN response. However, our data are not easily reconciled with this model. We provide genetic evidence that two RNA-sensing proteins, RIG-I and MDA5, participate in the IFN response to L. pneumophila. Importantly, these sensors do not seem to be required for the IFN response to L. pneumophila DNA, whereas we found that RIG-I was required for the response to L. pneumophila RNA. Thus, we hypothesize that bacterial RNA, or perhaps an induced host RNA, is the primary stimulus inducing the IFN response to L. pneumophila. Our study also identified a secreted effector protein, SdhA, as a key suppressor of the IFN response to L. pneumophila. Although viral suppressors of cytosolic RNA-sensing pathways have been previously identified, analogous bacterial factors have not been described. Thus, our results provide new insights into the molecular mechanisms by which an intracellular bacterial pathogen activates and also represses innate immune responses.

  11. Cytosolic SYT/SS18 isoforms are actin-associated proteins that function in matrix-specific adhesion.

    Directory of Open Access Journals (Sweden)

    Jaehong Kim

    2009-07-01

    Full Text Available SYT (SYnovial sarcoma Translocated gene or SS18 is widely produced as two isoforms, SYT/L and SYT/S, that are thought to function in the nucleus as transcriptional coactivators. Using isoform-specific antibodies, we detected a sizable pool of SYT isoforms in the cytosol where the proteins were organized into filamentous arrays. Actin and actin-associated proteins co-immunoprecipitated with SYT isoforms, which also co-sedimented and co-localized with the actin cytoskeleton in cultured cells and tissues. The association of SYT with actin bundles was extensive yet stopped short of the distal ends at focal adhesions. Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol. RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles. Furthermore, ablation of SYT led to markedly impaired adhesion and spreading on fibronectin and laminin-111 but not on collagen types I or IV. These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices.

  12. Cancer and molecular biomarkers of phase 2

    DEFF Research Database (Denmark)

    Dalhoff, Kim; Enghusen Poulsen, Henrik

    2005-01-01

    Associations between genotypes of phase 2 enzymes and cancer risk are extracted from epidemiological studies, namely case-control studies. Variant alleles in glutathione S-transferase (GST), UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), and N-acetyltransferase (NAT) have been used...... as molecular genetic biomarkers of risk. GSTM(my)1 has been associated with an increased risk of colorectal cancer, lung cancer, and bladder cancer and GSTP(pi)1 with prostate cancer. UGT1A1*28 and *37 are both associated with an increased risk of breast cancer as is SULT1A1*2. The presence of UGT1A1......*28 results in an increased risk of ovarian cancer and NAT2 of colorectal and lung cancer. A high frequency of SULT1A1*1 has been identified in patients with breast cancer; the role in colorectal cancer is more controversial. This chapter discusses the balance between carcinogen activation and detoxification...

  13. Studies on glucose-metabolizing enzymes in cytosolic and bacteroidal fractions of mungbean (Vigna radiata L.) and lentil (Lens culinaris L.) nodules.

    Science.gov (United States)

    Munjral, N; Gupta, A K; Kaur, N

    2007-06-01

    Nitrogen is exported in the form of ureides or amides from the nodules in pulse crops. In order to understand the carbon metabolism in ureide and amide exporting nodules, activities of enzymes involved in glucose metabolism were compared in cytosolic and bacteroidal fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules during development. Activities of hexokinase, fructokinase, phosphoglucomutase, fructose-1,6-bisphosphatase, phosphohexose isomerase and UDP-glucose pyrophosphorylase were detected in cytosolic fraction of nodules of both the crops during development. Out of these enzymes, specific activity of phosphohexose isomerase was the highest in nodules of both the crops, in comparison with other enzymes. In comparison with mungbean, activities of various enzymes were less in cytosolic fraction of lentil. Activities of hexokinase, fructokinase, phosphoglucomutase were present only in cytosolic fraction of mungbean (Vigna radiata L.), however, low activity of these enzymes was also observed in lentil (Lens culinaris L.) bacteroids. Activities of phosphohexose isomerase and fructose-1,6-bisphosphatase were higher in bacteroids of lentil, as compared to mungbean during early nodule development, but this pattern was reversed with progress of crop development. Higher activities of phosphoglucomutase and fructose-1,6-phosphatase in mungbean cytosolic fraction could lead to increased flow of carbon towards pentose phosphate pathway.

  14. Restricting glutamylcysteine synthetase activity to the cytosol or glutathione biosynthesis to the plastid is sufficient for normal plant development and stress tolerance.

    Science.gov (United States)

    Lim, B; Pasternak, M; Meyer, A J; Cobbett, C S

    2014-01-01

    The tripeptide glutathione (GSH) is an important metabolite with a broad spectrum of functions, and its homeostasis is essential to maintain cellular redox poise and effective responses to stress in plants. In Arabidopsis GSH is synthesised in two successive enzymatic steps by γ-glutamylcysteine synthetase (GSH1), localised exclusively in plastids, forming the pathway intermediate γ-glutamylcysteine (γ-EC), and then by glutathione synthetase (GSH2), which is located in both plastids and cytosol. This suggests a mechanism for γ-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is speculated that the cytosol may be the main compartment for GSH biosynthesis. With the availability of knockout lethal mutants of GSH1 and GSH2 in Arabidopsis, we were able to manipulate the GSH biosynthetic pathway within cells through transgenic techniques. We successfully complemented the gsh1 and gsh2 null mutants with a cytosol-targeted bacterial EcGSHA and plastid-targeted Arabidopsis GSH2 protein, respectively, to wild-type phenotypes. These transgenics were little affected under heavy metal (cadmium) or oxidative stress (H2 O2 ) when compared to the wild type. Collectively, our data show that redirecting GSH1 activity exclusively to the cytosol or restricting GSH biosynthesis to the plastids has no significant impact on development or stress resistance, suggesting efficient exchange of γ-EC and GSH between the plastid and cytosol compartments within cells. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  15. Surface-Enhanced Raman Spectroscopy (SERS Tracking of Chelerythrine, a Na+/K+ Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Kevin M. Dorney

    2013-12-01

    Full Text Available Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA chelerythrine (CET is a pro-apoptotic drug and Na+/K+ pump (NKP inhibitor in human lens epithelial cells (HLECs. In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS. Methods: Silver nanoparticles (AgNPs prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+ accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

  16. Surface-enhanced Raman spectroscopy (SERS) tracking of chelerythrine, a Na(+)/K(+) pump inhibitor, into cytosol and plasma membrane fractions of human lens epithelial cell cultures.

    Science.gov (United States)

    Dorney, Kevin M; Sizemore, Ioana E P; Alqahtani, Tariq; Adragna, Norma C; Lauf, Peter K

    2013-01-01

    The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na(+)/K(+) pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm(-1) marker band as a function of CET concentration. SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET(+)) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect. © 2014 S. Karger AG, Basel.

  17. Involvement of ABA- and H2O2-dependent cytosolic glucose-6-phosphate dehydrogenase in maintaining redox homeostasis in soybean roots under drought stress.

    Science.gov (United States)

    Wang, Huahua; Yang, Lidan; Li, Yan; Hou, Junjie; Huang, Junjun; Liang, Weihong

    2016-10-01

    The roles of abscisic acid (ABA) and hydrogen peroxide (H2O2) in inducing glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activity and the possible roles of G6PDH in regulating ascorbate-glutathione (AsA-GSH) cycle were investigated in soybean (Glycine max L.) roots under drought stress. Drought caused a marked increase of the total and cytosolic G6PDH activities and triggered a rapid ABA and H2O2 accumulation in soybean roots. Exogenous ABA or H2O2 treatment elevated the total and cytosolic G6PDH activities, whereas suppressing ABA or H2O2 production inhibited the drought-induced increase in total and cytosolic G6PDH activities, suggesting that ABA and H2O2 are required for drought-induced increase of total G6PDH activity, namely cytosolic G6PDH activity. Furthermore, ABA induced H2O2 production by stimulating NADPH oxidase activity under drought stress. Moreover, drought significantly increased the contents of AsA and GSH and the activities of key enzymes in AsA-GSH cycle, while application of G6PDH inhibitor to seedlings significantly reduced the above effect induced by drought. Taken together, these results indicate that H2O2 acting as a downstream signaling molecule of ABA mediates drought-induced increase in cytosolic G6PDH activity, and that enhanced cytosolic G6PDH activity maintains cellular redox homeostasis by regulating AsA-GSH cycle in soybean roots. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  18. Single molecular image of cytosolic free Ca2+ of skeletal muscle cells in rats pre- and post-exercise-induced fatigue

    Science.gov (United States)

    Liu, Yi; Zhang, Heming; Zhao, Yanping; Liu, Zhiming

    2009-08-01

    A growing body of literature indicated the cytosolic free Ca2+ concentration of skeletal muscle cells changes significantly during exercise-induced fatigue. But it is confusing whether cytosolic free Ca2+ concentration increase or decrease. Furthermore, current researches mainly adopt muscle tissue homogenate as experiment material, but the studies based on cellular and subcellular level is seldom. This study is aimed to establish rat skeletal muscle cell model of exercise-induced fatigue, and confirm the change of cytosolic free Ca2+ concentration of skeletal muscle cells in rats preand post- exercise-induced fatigue. In this research, six male Wistar rats were randomly divided into two groups: control group (n=3) and exercise-induced fatigue group (n=3). The former group were allowed to freely move and the latter were forced to loaded swimming to exhaustive. Three days later, all the rats were sacrificed, the muscle tissue from the same site of skeletal muscle were taken out and digested to cells. After primary culture of the two kinds of skeletal muscle cells from tissue, a fluorescent dye-Fluo-3 AM was used to label the cytosolic free Ca2+. The fluorescent of Ca2+ was recorded by confocal laser scanning microscopy. The results indicated that, the Ca2+ fluorescence intensity of cells from the rat of exercise-induced fatigue group was significantly higher than those in control group. In conclusion, cytosolic free Ca2+ concentration of skeletal muscle cells has a close relation with exercise-induced fatigue, and the increase of cytosolic free Ca2+ concentration may be one of the important factors of exercise-induced fatigue.

  19. Phosphorylation of the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor is required for the interaction with a cytosolic protein

    DEFF Research Database (Denmark)

    Rosorius, O; Issinger, O G; Braulke, T

    1993-01-01

    ) and to 35- and 91-kDa proteins salt-washed from bovine brain membranes. Gel filtration suggested that TIP 35 is part of a higher molecular mass complex of approximately 130-150 kDa. Inhibition studies, using non-phosphorylated and phosphorylated MPR 300-CT and cross-linking, indicate that the interaction...... with a cytosolic protein depending on the phosphorylation by a casein kinase II-like kinase. The cross-linking with salt-washed membrane proteins, however, is inhibited by non-phosphorylated MPR 300-CT, suggesting that different structural determinants in the MPR 300-CT interact with cytosol- and membrane...

  20. Cytosol protein regulation in H295R steroidogenesis model induced by the zearalenone metabolites, α- and β-zearalenol.

    Science.gov (United States)

    Busk, Øyvind L; Frizzell, Caroline; Verhaegen, Steven; Uhlig, Silvio; Connolly, Lisa; Ropstad, Erik; Sørlie, Morten

    2012-01-01

    α- and β-zearalenol (α-ZOL and β-ZOL, respectively) are metabolites of the mycotoxin zearalenone (ZEN). All three individual mycotoxins have shown to be biological active i.e. being estrogenic and able to stimulate cellular proliferation albeit at different strengths. In this work, cytosol protein expression was determined by using stable-isotope labelling by amino acids in cell culture (SILAC) upon exposure of α-ZOL and β-ZOL to the steroidogenesis cell model H295R. A total of 14 and 5 individual proteins were found to be significantly regulated by α-ZOL and β-ZOL, respectively. Interestingly, there were no common protein regulations by the metabolites or the parent mycotoxin ZEN. Furthermore, the regulated proteins were assigned to networks and groups of actions that also differed from one another suggesting that the three individual mycotoxins may have unique biological activities. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    Science.gov (United States)

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-07-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

  2. Cytosolic RNA:DNA Duplexes Generated by Endogenous Reverse Transcriptase Activity as Autonomous Inducers of Skin Inflammation in Psoriasis.

    Science.gov (United States)

    Molès, Jean-Pierre; Griez, Anthony; Guilhou, Jean-Jacques; Girard, Céline; Nagot, Nicolas; Van de Perre, Philippe; Dujols, Pierre

    2017-01-01

    Psoriasis is a chronic skin disease of unknown ætiology. Recent studies suggested that a large amount of cytosolic DNA (cyDNA) in keratinocytes is breaking keratinocytes DNA tolerance and promotes self-sustained inflammation in the psoriatic lesion. We investigated the origin of this cyDNA. We show that, amongst all the possible DNA structures, the cyDNA could be present as RNA:DNA duplexes in keratinocytes. We further show that endogenous reverse transcriptase activities generate such duplexes and consequently activate the production of Th1-inflammatory cytokines. These observations open a new research avenue related to endogenous retroelements for the aetiology of psoriasis and probably of other human chronic inflammatory diseases.

  3. The Arabidopsis thalianaK+-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre

    KAUST Repository

    Al-Younis, Inas

    2015-11-27

    Adenylate Cyclases (ACs) catalyze the formation of the second messenger cyclic adenosine 3′, 5′-monophosphate (cAMP) from adenosine 5’-triphosphate (ATP). Although cAMP is increasingly recognized as an important signaling molecule in higher plants, ACs have remained somewhat elusive. Here we used a search motif derived from experimentally tested guanylyl cyclases (GCs), substituted the residues essential for substrate specificity and identified the Arabidopsis thaliana K+-uptake permease 7 (AtKUP7) as one of several candidate ACs. Firstly, we show that a recombinant N-terminal, cytosolic domain of AtKUP71-100 is able to complement the AC-deficient mutant cyaA in Escherichia coli and thus restoring the fermentation of lactose, and secondly, we demonstrate with both enzyme immunoassays and mass spectrometry that a recombinant AtKUP71-100 generates cAMP in vitro.

  4. Graphene nanoplatelets spontaneously translocate into the cytosol and physically interact with cellular organelles in the fish cell line PLHC-1

    Energy Technology Data Exchange (ETDEWEB)

    Lammel, Tobias; Navas, José M., E-mail: jmnavas@inia.es

    2014-05-01

    Highlights: • We assessed the cytotoxicity and uptake of graphene nanomaterials in PLHC-1 cells. • GO and CXYG nanoplatelets caused physical injury of the plasma membrane. • GO and CXYG accumulated in the cytosol and interacted with cellular organelles. • PLHC-1 cells exposed to GO/CXYG demonstrated high ROS levels but low cytotoxicity. • ROS formation was related with GO/CXYG-induced structural damage of mitochondria. - Abstract: Graphene and graphene derivatives constitute a novel class of carbon-based nanomaterials being increasingly produced and used in technical and consumer applications. Release of graphene nanoplatelets during the life cycle of these applications may result in human and environmental exposure calling for assessment of their potential to cause harm to humans and wildlife. This study aimed to assess the toxicity of graphene oxide (GO) and carboxyl graphene (CXYG) nanoplatelets to non-mammalian species using the fish cell line PLHC-1 as in vitro model. The cytotoxicity of GO and CXYG was assessed using different assays measuring alterations in plasma membrane integrity, metabolic activity, and lysosomal and mitochondrial function. The induction of oxidative stress was assessed by measuring intracellular reactive oxygen species (ROS) levels. Interaction with the plasma membrane and internalization of nanoplatelets were investigated by electron microscopy. Graphene nanoplatelets spontaneously penetrated through the plasma membrane and accumulated in the cytosol, where they further interacted with mitochondrial and nuclear membranes. PLHC-1 cells demonstrated significantly reduced mitochondrial membrane potential (MMP) and increased ROS levels at 16 μg/ml GO and CXYG (72 h), but barely any decrease in cell viability. The observation of intracellular graphene accumulations not enclosed by membranes suggests that GO and CXYG internalization in fish hepatoma cells occurs through an endocytosis-independent mechanism.

  5. Despite differences in cytosolic calcium regulation, lidocaine toxicity is similar in adult and neonatal rat dorsal root ganglia in vitro.

    Science.gov (United States)

    Doan, Lisa V; Eydlin, Olga; Piskoun, Boris; Kline, Richard P; Recio-Pinto, Esperanza; Rosenberg, Andrew D; Blanck, Thomas J J; Xu, Fang

    2014-01-01

    Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action of local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, the authors examined whether there were any differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons. DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine. The mean KCl-induced calcium transient was greater in P7 neurons (P lidocaine significantly inhibited KCl-induced calcium responses in both ages (P lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly, cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages. Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.

  6. Despite Differences in Cytosolic Calcium Regulation, Lidocaine Toxicity Is Similar in Adult and Neonatal Rat Dorsal Root Ganglia in Vitro

    Science.gov (United States)

    Doan, Lisa V.; Eydlin, Olga; Piskoun, Boris; Kline, Richard P; Recio-Pinto, Esperanza; Rosenberg, Andrew D; Blanck, Thomas JJ; Xu, Fang

    2013-01-01

    Background Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action for local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, we examined whether there were differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons. Methods DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine. Results The mean KCl-induced calcium transient was greater in P7 neurons (p lidocaine significantly inhibited KCl-induced calcium responses in both ages (p lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages. Conclusions Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses. PMID:23851347

  7. Excessive cytosolic DNA fragments as a potential trigger of Graves’ disease: an encrypted message sent by animal models

    Directory of Open Access Journals (Sweden)

    Yuqian Luo

    2016-11-01

    Full Text Available Graves’ hyperthyroidism is caused by autoantibodies directed against the thyroid stimulating hormone receptor (TSHR that mimic the action of TSH. The establishment of Graves’ hyperthyroidism in experimental animals has proven to be an important approach to dissect the mechanisms of self-tolerance breakdown that lead to the production of thyroid-stimulating TSHR autoantibodies (TSAbs. ‘Shimojo’s model was the first successful Graves’ animal model, wherein immunization with fibroblasts cells expressing TSHR and a major histocompatibility complex (MHC class II molecule, but not either alone, induced TSAb production in AKR/N (H-2k mice. This model highlights the importance of coincident MHC class II expression on TSHR-expressing cells in the development of Graves’ hyperthyroidism. These data are also in agreement with the observation that Graves’ thyrocytes often aberrantly express MHC class II antigens via mechanisms that remain unclear. Our group demonstrated that cytosolic self-genomic DNA fragments derived from sterile injured cells can induce aberrant MHC class II expression and production of multiple inflammatory cytokines and chemokines in thyrocytes in vitro, suggesting that severe cell injury may initiate immune responses in a way that is relevant to thyroid autoimmunity mediated by cytosolic DNA signaling. Furthermore, more recent successful Graves’ animal models were primarily established by immunizing mice with TSHR-expressing plasmids or adenovirus. In these models, double-stranded DNA vaccine contents presumably exert similar immune-activating effect in cells at inoculation sites and thus might pave the way toward successful Graves’ animal models. This review focuses on evidence suggesting that cell injury-derived self-DNA fragments could act as Graves’ disease triggers.

  8. High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB-REDO strategies

    Energy Technology Data Exchange (ETDEWEB)

    Rimsa, Vadim; Eadsforth, Thomas C. [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Joosten, Robbie P. [Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam (Netherlands); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom)

    2014-02-01

    The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase. A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB-REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn{sup 2+}-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn{sup 2+}, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.

  9. Prediction of human drug clearance by multiple metabolic pathways: integration of hepatic and intestinal microsomal and cytosolic data.

    Science.gov (United States)

    Cubitt, Helen E; Houston, J Brian; Galetin, Aleksandra

    2011-05-01

    The current study assesses hepatic and intestinal glucuronidation, sulfation, and cytochrome P450 (P450) metabolism of raloxifene, quercetin, salbutamol, and troglitazone using different in vitro systems. The fraction metabolized by conjugation and P450 metabolism was estimated in liver and intestine, and the importance of multiple metabolic pathways on accuracy of clearance prediction was assessed. In vitro intrinsic sulfation clearance (CL(int, SULT)) was determined in human intestinal and hepatic cytosol and compared with hepatic and intestinal microsomal glucuronidation (CL(int, UGT)) and P450 clearance (CL(int, CYP)) expressed per gram of tissue. Hepatic and intestinal cytosolic scaling factors of 80.7 mg/g liver and 18 mg/g intestine were estimated from published data. Scaled CL(int, SULT) ranged between 0.7 and 11.4 ml · min(-1) · g(-1) liver and 0.1 and 3.3 ml · min(-1) · g(-1) intestine (salbutamol and quercetin were the extremes). Salbutamol was the only compound with a high extent of sulfation (51 and 28% of total CL(int) for liver and intestine, respectively) and also significant renal clearance (26-57% of observed plasma clearance). In contrast, the clearance of quercetin was largely accounted for by glucuronidation. Drugs metabolized by multiple pathways (raloxifene and troglitazone) demonstrated improved prediction of intravenous clearance using data from all hepatic pathways (44-86% of observed clearance) compared with predictions based only on the primary pathway (22-36%). The assumption of no intestinal first pass resulted in underprediction of oral clearance for raloxifene, troglitazone, and quercetin (3-22% of observed, respectively). Accounting for the intestinal contribution to oral clearance via estimated intestinal availability improved prediction accuracy for raloxifene and troglitazone (within 2.5-fold of observed). Current findings emphasize the importance of both hepatic and intestinal conjugation for in vitro-in vivo extrapolation

  10. Excessive Cytosolic DNA Fragments as a Potential Trigger of Graves' Disease: An Encrypted Message Sent by Animal Models.

    Science.gov (United States)

    Luo, Yuqian; Yoshihara, Aya; Oda, Kenzaburo; Ishido, Yuko; Suzuki, Koichi

    2016-01-01

    Graves' hyperthyroidism is caused by autoantibodies directed against the thyroid-stimulating hormone receptor (TSHR) that mimic the action of TSH. The establishment of Graves' hyperthyroidism in experimental animals has proven to be an important approach to dissect the mechanisms of self-tolerance breakdown that lead to the production of thyroid-stimulating TSHR autoantibodies (TSAbs). "Shimojo's model" was the first successful Graves' animal model, wherein immunization with fibroblasts cells expressing TSHR and a major histocompatibility complex (MHC) class II molecule, but not either alone, induced TSAb production in AKR/N (H-2k) mice. This model highlights the importance of coincident MHC class II expression on TSHR-expressing cells in the development of Graves' hyperthyroidism. These data are also in agreement with the observation that Graves' thyrocytes often aberrantly express MHC class II antigens via mechanisms that remain unclear. Our group demonstrated that cytosolic self-genomic DNA fragments derived from sterile injured cells can induce aberrant MHC class II expression and production of multiple inflammatory cytokines and chemokines in thyrocytes in vitro, suggesting that severe cell injury may initiate immune responses in a way that is relevant to thyroid autoimmunity mediated by cytosolic DNA signaling. Furthermore, more recent successful Graves' animal models were primarily established by immunizing mice with TSHR-expressing plasmids or adenovirus. In these models, double-stranded DNA vaccine contents presumably exert similar immune-activating effect in cells at inoculation sites and thus might pave the way toward successful Graves' animal models. This review focuses on evidence suggesting that cell injury-derived self-DNA fragments could act as Graves' disease triggers.

  11. Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brat Dawid

    2012-09-01

    Full Text Available Abstract Background The branched chain alcohol isobutanol exhibits superior physicochemical properties as an alternative biofuel. The yeast Saccharomyces cerevisiae naturally produces low amounts of isobutanol as a by-product during fermentations, resulting from the catabolism of valine. As S. cerevisiae is widely used in industrial applications and can easily be modified by genetic engineering, this microorganism is a promising host for the fermentative production of higher amounts of isobutanol. Results Isobutanol production could be improved by re-locating the valine biosynthesis enzymes Ilv2, Ilv5 and Ilv3 from the mitochondrial matrix into the cytosol. To prevent the import of the three enzymes into yeast mitochondria, N-terminally shortened Ilv2, Ilv5 and Ilv3 versions were constructed lacking their mitochondrial targeting sequences. SDS-PAGE and immunofluorescence analyses confirmed expression and re-localization of the truncated enzymes. Growth tests or enzyme assays confirmed enzymatic activities. Isobutanol production was only increased in the absence of valine and the simultaneous blockage of the mitochondrial valine synthesis pathway. Isobutanol production could be even more enhanced after adapting the codon usage of the truncated valine biosynthesis genes to the codon usage of highly expressed glycolytic genes. Finally, a suitable ketoisovalerate decarboxylase, Aro10, and alcohol dehydrogenase, Adh2, were selected and overexpressed. The highest isobutanol titer was 0.63 g/L at a yield of nearly 15 mg per g glucose. Conclusion A cytosolic isobutanol production pathway was successfully established in yeast by re-localization and optimization of mitochondrial valine synthesis enzymes together with overexpression of Aro10 decarboxylase and Adh2 alcohol dehydrogenase. Driving forces were generated by blocking competition with the mitochondrial valine pathway and by omitting valine from the fermentation medium. Additional deletion of

  12. Pomegranate juice inhibits sulfoconjugation in Caco-2 human colon carcinoma cells.

    Science.gov (United States)

    Saruwatari, Ayako; Okamura, Shigeaki; Nakajima, Yoko; Narukawa, Yuji; Takeda, Tadahiro; Tamura, Hiroomi

    2008-12-01

    Several fruit juices have been reported to cause food-drug interactions, mainly affecting cytochrome P450 activity; however, little is known about the effects of fruit juices on conjugation reactions. Among several fruit juices tested (apple, peach, orange, pineapple, grapefruit, and pomegranate), pomegranate juice potently inhibited the sulfoconjugation of 1-naphthol in Caco-2 cells. This inhibition was both dose- and culture time-dependent, with a 50% inhibitory concentration (IC(50)) value calculated at 2.7% (vol/vol). In contrast, no obvious inhibition of glucuronidation of 1-naphthol in Caco-2 cells was observed by any of the juices examined. Punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in Caco-2 cells with an IC(50) of 45 microM, which is consistent with that of pomegranate juice. These data suggest that punicalagin is mainly responsible for the inhibition of sulfoconjugation by pomegranate juice. We additionally demonstrated that pomegranate juice and punicalagin both inhibit phenol sulfotransferase activity in Caco-2 cells in vitro, at concentrations that are almost equivalent to those used in the Caco-2 cells. Pomegranate juice, however, shows no effects on the expression of the sulfotransferase SULT1A family of genes (SULT1A1 and SULT1A3) in Caco-2 cells. These results indicate that the inhibition of sulfotransferase activity by punicalagin in Caco-2 cells is responsible for the reductions seen in 1-naphthyl sulfate accumulation. Our data also suggest that constituents of pomegranate juice, most probably punicalagin, impair the enteric functions of sulfoconjugation and that this might have effects upon the bioavailability of drugs and other compounds present in food and in the environment. These effects might be related to the anticarcinogenic properties of pomegranate juice.

  13. Cytosolic glutamine synthetase Gln1;2 is the main isozyme contributing to GS1 activity and can be up-regulated to relieve ammonium toxicity

    DEFF Research Database (Denmark)

    Guan, Miao; de Bang, Thomas Christian; Pedersen, Carsten

    2016-01-01

    Cytosolic GS1 (Gln synthetase) is central for ammonium assimilation in plants. High ammonium treatment enhanced the expression of the GS1 isogene Gln-1;2 encoding a low-affinity high-capacity GS1 protein in Arabidopsis (Arabidopsis thaliana) shoots. Under the same conditions, the expression of th...

  14. Evaluation of the kinetics of beta-elimination reactions of selenocysteine Se-conjugates in human renal cytosol : possible implications for the use as kidney selective prodrugs

    NARCIS (Netherlands)

    Rooseboom, M; Vermeulen, N P; Andreadou, I; Commandeur, J N

    This study was performed to evaluate whether selenocysteine Se-conjugates are substrates for human cysteine conjugate beta-lyase enzymes. By testing kidney cytosols of three different humans, we studied interindividual differences in beta-lyase enzymes in humans. A series of 22 selenocysteine

  15. Cytosolic triglycerides and oxidative stress in central obesity : the missing link between excessive atherosclerosis, endothelial dysfunction, and beta-cell failure?

    NARCIS (Netherlands)

    Bakker, SJL; IJzerman, RG; Teerlink, T; Westerhoff, HV; Gans, ROB; Heine, RJ

    Central obesity is increasingly recognized as a risk factor for atherosclerosis and type 2 diabetes mellitus. Here we present a hypothesis that may explain the excess atherosclerosis, endothelial dysfunction and progressive beta-cell failure. Central obesity is associated with increased cytosolic

  16. Acute toxicity of a shoreline cleaner, CytoSol, mixed with oil and ecological risk assessment of its use on the Galician Coast.

    Science.gov (United States)

    Rial, Diego; Beiras, Ricardo; Vázquez, José A; Murado, Miguel A

    2010-10-01

    The application of embryo-larval bioassay with the purple sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis at 48 hours, and with neonates of the mysid Siriella armata at 96 hours, was used to evaluate the acute toxicities of the following preparations: (1) the shoreline cleaning agent CytoSol; (2) the water-accommodated fraction of CytoSol plus a light crude oil; and (3) the runoff from a pilot-scale treatment with CytoSol of a rocky coastal substrate impregnated with residues from the Prestige oil spill (which occurred on November 19, 2002). The mussel was the most sensitive organism to CytoSol and runoff effects (EC(50) = 8.0 microL/L and 64.3 mL/L, respectively), and the mysid was the least sensitive to the runoff (EC(50) > 200 mL/L). The predicted no-effect environmental concentration (PNEC) was calculated from the no observed-effect concentration of the species most sensitive to the runoff. The predicted environmental concentration (PEC) was estimated from a simple and reasonable dilution model, and the PEC/PNEC ratio was calculated according to the area treated and the values of the variables considered in the model. Implications for the management of the treatment operations are discussed.

  17. Steady-state distribution of metals among metallothionein and other cytosolic ligands and links to cytotoxicity in bivalves living along a polymetallic gradient

    Energy Technology Data Exchange (ETDEWEB)

    Giguere, Anik; Couillard, Yves; Campbell, Peter G.C.; Perceval, Olivier; Hare, Landis; Pinel-Alloul, Bernadette; Pellerin, Jocelyne

    2003-07-16

    The present study was designed to assess the environmental effects of metals in a field setting. We explored exposure{yields}bioaccumulation{yields}effects relationships in freshwater molluscs exposed to metals in their natural habitat. Indigenous floater mussels (Pyganodon grandis) were collected from ten limnologically similar lakes located along a Cd, Cu and Zn gradient. Ambient free-metal ion concentrations were estimated as a measure of metal exposure. Metallothionein (MT) was measured in mussel gills and metal partitioning among the various cytosolic protein pools was determined by size exclusion chromatography. Various biomarkers were also measured, including malondialdehyde (MDA) concentrations in the gills and in the digestive gland, glutathione-peroxidase and glutathione-reductase activities in the digestive gland, and lipid concentrations in the gonad. Cadmium and MT concentrations in the gill cytosol increased along the contamination gradient, but Cu and Zn levels were independent of the ambient free-metal ion concentrations. The distribution of Cd among the various cytosolic complexes remained quite constant: 80% in the MT-like pool, 7% in the low molecular weight pool (LMW<1.8 kDa) and 13% in the high molecular weight pool (HMW>18 kDa). For these chronically exposed molluscs there was thus no threshold exposure concentration above which spillover of Cd occurred from the MT pool to other cytosolic ligands. However, the presence of Cd in the LMW and HMW fractions suggests that metal detoxification was imperfect, i.e. that P. grandis was subject to some Cd-related stress at low chronic exposure concentrations. Consistent with this suggestion, MDA concentrations, an indicator of oxidative stress, increased with gill cytosolic Cd. In the digestive gland, MDA concentrations were unrelated to any of the measured metals, but glutathione-peroxidase and glutathione-reductase activities increased with gill cytosolic copper. We speculate that cytosolic Cu

  18. Apoptotic DNA Degradation into Oligonucleosomal Fragments, but Not Apoptotic Nuclear Morphology, Relies on a Cytosolic Pool of DFF40/CAD Endonuclease*

    Science.gov (United States)

    Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Gabernet, Gisela; García-Belinchón, Mercè; Sánchez-Osuna, María; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.

    2012-01-01

    Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. PMID:22253444

  19. Bacteria modulate the CD8+ T cell epitope repertoire of host cytosol-exposed proteins to manipulate the host immune response.

    Directory of Open Access Journals (Sweden)

    Yaakov Maman

    2011-10-01

    Full Text Available The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested.

  20. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Veenhuis Marten

    2005-10-01

    Full Text Available Abstract Background Native as well as recombinant bacterial cell surface layer (S-layer protein of Geobacillus (G. stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099 of the S-layer protein and EGFP (enhanced green fluorescent protein, in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. Results Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S. cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE promoter. Conclusion The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of

  1. Initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa related to the human cytosolic 5'-nucleotidase I.

    Science.gov (United States)

    Santos, Clelton A; Saraiva, Antonio M; Toledo, Marcelo A S; Beloti, Lilian L; Crucello, Aline; Favaro, Marianna T P; Horta, Maria A C; Santiago, André S; Mendes, Juliano S; Souza, Alessandra A; Souza, Anete P

    2013-01-01

    The 5'-nucleotidases constitute a ubiquitous family of enzymes that catalyze either the hydrolysis or the transfer of esterified phosphate at the 5' position of nucleoside monophosphates. These enzymes are responsible for the regulation of nucleotide and nucleoside levels in the cell and can interfere with the phosphorylation-dependent activation of nucleoside analogs used in therapies targeting solid tumors and viral infections. In the present study, we report the initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa that is related to the human cytosolic 5'-nucleotidase I. X. fastidiosa is a plant pathogenic bacterium that is responsible for numerous economically important crop diseases. Biochemical assays confirmed the phosphatase activity of the recombinant purified enzyme and revealed metal ion dependence for full enzyme activity. In addition, we investigated the involvement of Xf5'-Nt in the formation of X. fastidiosa biofilms, which are structures that occlude the xylem vessels of susceptible plants and are strictly associated with bacterial pathogenesis. Using polyclonal antibodies against Xf5'-Nt, we observed an overexpression of Xf5'-Nt during the initial phases of X. fastidiosa biofilm formation that was not observed during X. fastidiosa planktonic growth. Our results demonstrate that the de/phosphorylation network catalyzed by 5'-nucleotidases may play an important role in bacterial biofilm formation, thereby contributing novel insights into bacterial nucleotide metabolism and pathogenicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Germ-Cell-Specific Inflammasome Component NLRP14 Negatively Regulates Cytosolic Nucleic Acid Sensing to Promote Fertilization.

    Science.gov (United States)

    Abe, Takayuki; Lee, Albert; Sitharam, Ramaswami; Kesner, Jordan; Rabadan, Raul; Shapira, Sagi D

    2017-04-18

    Cytosolic sensing of nucleic acids initiates tightly regulated programs to limit infection. Oocyte fertilization represents a scenario wherein inappropriate responses to exogenous yet non-pathogen-derived nucleic acids would have negative consequences. We hypothesized that germ cells express negative regulators of nucleic acid sensing (NAS) in steady state and applied an integrated data-mining and functional genomics approach to identify a rheostat of DNA and RNA sensing-the inflammasome component NLRP14. We demonstrated that NLRP14 interacted physically with the nucleic acid sensing pathway and targeted TBK1 (TANK binding kinase 1) for ubiquitination and degradation. We further mapped domains in NLRP14 and TBK1 that mediated the inhibitory function. Finally, we identified a human nonsense germline variant associated with male sterility that results in loss of NLRP14 function and hyper-responsiveness to nucleic acids. The discovery points to a mechanism of nucleic acid sensing regulation that may be of particular importance in fertilization. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Inhibition of Anthracycline Alcohol Metabolite Formation in Human Heart Cytosol: A Potential Role for Several Promising Drugs.

    Science.gov (United States)

    Mordente, Alvaro; Silvestrini, Andrea; Martorana, Giuseppe Ettore; Tavian, Daniela; Meucci, Elisabetta

    2015-11-01

    The clinical efficacy of anthracyclines (e.g., doxorubicin and daunorubicin) in cancer therapy is limited by their severe cardiotoxicity, the etiology of which is still not fully understood. The development of anthracycline-induced cardiomyopathy has been found to correlate with myocardial formation and accumulation of anthracycline secondary alcohol metabolites (e.g., doxorubicinol and daunorubicinol) that are produced by distinct cytosolic NADPH-dependent reductases. The aim of the current study is to identify chemical compounds capable of inhibiting myocardial reductases implied in anthracycline reductive metabolism in an attempt to decrease the production of cardiotoxic C-13 alcohol metabolites. Among the variety of tested compounds (metal chelators, radical scavengers, antioxidants, β-blockers, nitrone spin traps, and lipid-lowering drugs), ebselen, cyclopentenone prostaglandins, nitric oxide donors, and short-chain coenzyme Q analogs resulted in being effective inhibitors of both doxorubicinol and daunorubicinol formation. In particular, ebselen (as well as ebselen diselenide, its storage form in the cells) was the most potent inhibitor of cardiotoxic anthracycline alcohol metabolites with 50% inhibition of doxorubicinol formation at 0.2 mol Eq of ebselen with respect to doxorubicin concentration. The high efficacy, together with its favorable pharmacological profile (low toxicity, lack of adverse effects, and metabolic stability) portends ebselen as a promising cardioprotective agent against anthracycline-induced cardiotoxicity. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Hepatitis C virus non-structural protein 3 interacts with cytosolic 5'(3'-deoxyribonucleotidase and partially inhibits its activity.

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    Chiu-Ping Fang

    Full Text Available Infection with hepatitis C virus (HCV is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3 is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5'(3'-deoxyribonucleotidase (cdN, dNT-1 was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

  5. Analyzing the effect of decreasing cytosolic triosephosphate isomerase on Solanum tuberosum hairy root cells using a kinetic-metabolic model.

    Science.gov (United States)

    Valancin, Alexandre; Srinivasan, Balasubrahmanyan; Rivoal, Jean; Jolicoeur, Mario

    2013-03-01

    A kinetic-metabolic model of Solanum tuberosum hairy roots is presented in the interest of understanding the effect on the plant cell metabolism of a 90% decrease in cytosolic triosephosphate isomerase (cTPI, EC 5.3.1.1) expression by antisense RNA. The model considers major metabolic pathways including glycolysis, pentose phosphate pathway, and TCA cycle, as well as anabolic reactions leading to lipids, nucleic acids, amino acids, and structural hexoses synthesis. Measurements were taken from shake flask cultures for six extracellular nutrients (sucrose, fructose, glucose, ammonia, nitrate, and inorganic phosphate) and 15 intracellular compounds including sugar phosphates (G6P, F6P, R5P, E4P) and organic acids (PYR, aKG, SUCC, FUM, MAL) and the six nutrients. From model simulations and experimental data it can be noted that plant cell metabolism redistributes metabolic fluxes to compensate for the cTPI decrease, leading to modifications in metabolites levels. Antisense roots showed increased exchanges between the pentose phosphate pathway and the glycolysis, an increased oxygen uptake and growth rate. Copyright © 2012 Wiley Periodicals, Inc.

  6. Plasma beads loaded with Candida albicans cytosolic proteins impart protection against the fungal infection in BALB/c mice.

    Science.gov (United States)

    Ahmad, Ejaj; Fatima, Munazza T; Saleemuddin, M; Owais, M

    2012-11-06

    The development of a prophylactic vaccine against systemic candidiasis, employing Candida albicans cytosolic proteins (Cp) as antigen and fibrin cross-linked plasma beads as an antigen bearing dual delivery system is described. Groups of mice were administered either with free Cp, or Cp entrapped in plasma beads, Cp entrapped in liposomes or liposome encapsulated Cp further entrapped in plasma beads. Humoral immunity was studied by measuring the anti-Cp antibody titers in the sera of the immunized animals. Induction of cell-mediated immunity was assessed by delayed type hypersensitivity (DTH), NO production, up-regulation of co-stimulatory molecules viz. CD80, CD86 on APCs on one hand and T-cells proliferation as well as induction of IFN-γ and IL-4 on the other. The efficacy of various vaccine formulations in protecting mice against a lethal challenge with C. albicans, was assessed by determining animal survival rate and fungal burden in the systemic circulation and vital organs. Among various Cp-based vaccines investigated, the preparation containing liposomized Cp entrapped in plasma beads imparted superior protection in the immunized mice as compared to other antigens delivery systems. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Increasing the carbohydrate storage capacity of plants by engineering a glycogen-like polymer pool in the cytosol.

    Science.gov (United States)

    Eicke, Simona; Seung, David; Egli, Barbara; Devers, Emanuel A; Streb, Sebastian

    2017-03-01

    Global demand for higher crop yields and for more efficient utilization of agricultural products will grow over the next decades. Here, we present a new concept for boosting the carbohydrate content of plants, by channeling photosynthetically fixed carbon into a newly engineered glucose polymer pool. We transiently expressed the starch/glycogen synthases from either Saccharomyces cerevisiae or Cyanidioschyzon merolae, together with the starch branching enzyme from C. merolae, in the cytosol of Nicotiana benthamiana leaves. This effectively built a UDP-glucose-dependent glycogen biosynthesis pathway. Glycogen synthesis was observed with Transmission Electron Microscopy, and the polymer structure was further analyzed. Within three days of enzyme expression, glycogen content of the leaf was 5-10 times higher than the starch levels of the control. Further, the leaves produced less starch and sucrose, which are normally the carbohydrate end-products of photosynthesis. We conclude that after enzyme expression, the newly fixed carbohydrates were routed into the new glycogen sink and trapped. Our approach allows carbohydrates to be efficiently stored in a new subcellular compartment, thus increasing the value of vegetative crop tissues for biofuel production or animal feed. The method also opens new potential for increasing the sink strength of heterotrophic tissues. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  8. Light-dependent regulation of ascorbate in tomato by a monodehydroascorbate reductase localized in peroxisomes and the cytosol.

    Science.gov (United States)

    Gest, Noé; Garchery, Cécile; Gautier, Hélène; Jiménez, Ana; Stevens, Rebecca

    2013-04-01

    Ascorbate is a powerful antioxidant in plants, and its levels are an important quality criteria in commercial species. Factors influencing these levels include environmental variations, particularly light, and the genetic control of its biosynthesis, recycling and degradation. One of the genes involved in the recycling pathway encodes a monodehydroascorbate reductase (MDHAR), an enzyme catalysing reduction of the oxidized radical of ascorbate, monodehydroascorbate, to ascorbate. In plants, MDHAR belongs to a multigene family. Here, we report the presence of an MDHAR isoform in both the cytosol and peroxisomes and show that this enzyme negatively regulates ascorbate levels in Solanum lycopersicum (tomato). Transgenic lines overexpressing MDHAR show a decrease in ascorbate levels in leaves, whereas lines where MDHAR is silenced show an increase in these levels in both fruits and leaves. Furthermore, the intensity of these differences is light dependent. The unexpected effect of this MDHAR on ascorbate levels cannot be explained by changes in the expression of Smirnoff-Wheeler pathway genes, or the activity of enzymes involved in degradation (ascorbate peroxidase) or recycling of ascorbate (dehydroascorbate reductase and glutathione reductase), suggesting a previously unidentified mechanism regulating ascorbate levels. © 2012 INRA Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  9. Proteomic Profiling of Cytosolic Glutathione Transferases from Three Bivalve Species: Corbicula fluminea, Mytilus galloprovincialis and Anodonta cygnea

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    José Carlos Martins

    2014-01-01

    Full Text Available Suspension-feeding bivalves are considered efficient toxin vectors with a relative insensitivity to toxicants compared to other aquatic organisms. This fact highlights the potential role of detoxification enzymes, such as glutathione transferases (GSTs, in this bivalve resistance. Nevertheless, the GST system has not been extensively described in these organisms. In the present study, cytosolic GSTs isoforms (cGST were surveyed in three bivalves with different habitats and life strategies: Corbicula fluminea, Anodonta cygnea and Mytilus galloprovincialis. GSTs were purified by glutathione-agarose affinity chromatography, and the collection of expressed cGST classes of each bivalve were identified using a proteomic approach. All the purified extracts were also characterized kinetically. Results reveal variations in cGST subunits collection (diversity and properties between the three tested bivalves. Using proteomics, four pi-class and two sigma-class GST subunits were identified in M. galloprovincialis. C. fluminea also yielded four pi-class and one sigma-class GST subunits. For A. cygnea, two mu-class and one pi-class GST subunits were identified, these being the first record of GSTs from these freshwater mussels. The affinity purified extracts also show differences regarding enzymatic behavior among species. The variations found in cGST collection and kinetics might justify diverse selective advantages for each bivalve organism.

  10. Proteomic profiling of cytosolic glutathione transferases from three bivalve species: Corbicula fluminea, Mytilus galloprovincialis and Anodonta cygnea.

    Science.gov (United States)

    Martins, José Carlos; Campos, Alexandre; Osório, Hugo; da Fonseca, Rute; Vasconcelos, Vítor

    2014-01-27

    Suspension-feeding bivalves are considered efficient toxin vectors with a relative insensitivity to toxicants compared to other aquatic organisms. This fact highlights the potential role of detoxification enzymes, such as glutathione transferases (GSTs), in this bivalve resistance. Nevertheless, the GST system has not been extensively described in these organisms. In the present study, cytosolic GSTs isoforms (cGST) were surveyed in three bivalves with different habitats and life strategies: Corbicula fluminea, Anodonta cygnea and Mytilus galloprovincialis. GSTs were purified by glutathione-agarose affinity chromatography, and the collection of expressed cGST classes of each bivalve were identified using a proteomic approach. All the purified extracts were also characterized kinetically. Results reveal variations in cGST subunits collection (diversity and properties) between the three tested bivalves. Using proteomics, four pi-class and two sigma-class GST subunits were identified in M. galloprovincialis. C. fluminea also yielded four pi-class and one sigma-class GST subunits. For A. cygnea, two mu-class and one pi-class GST subunits were identified, these being the first record of GSTs from these freshwater mussels. The affinity purified extracts also show differences regarding enzymatic behavior among species. The variations found in cGST collection and kinetics might justify diverse selective advantages for each bivalve organism.

  11. Cytosolic guanine nucledotide binding deficient form of transglutaminase 2 (R580a potentiates cell death in oxygen glucose deprivation.

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    Gozde Colak

    Full Text Available Transglutaminase 2 (TG2 is a hypoxia-responsive protein that is a calcium-activated transamidating enzyme, a GTPase and a scaffolding/linker protein. Upon activation TG2 undergoes a large conformational change, which likely affects not only its enzymatic activities but its non-catalytic functions as well. The focus of this study was on the role of transamidating activity, conformation and localization of TG2 in ischemic cell death. Cells expressing a GTP binding deficient form of TG2 (TG2-R580A with high basal transamidation activity and a more extended conformation showed significantly increased cell death in response to oxygen-glucose deprivation; however, targeting TG2-R580A to the nucleus abrogated its detrimental role in oxygen-glucose deprivation. Treatment of cells expressing wild type TG2, TG2-C277S (a transamidating inactive mutant and TG2-R580A with Cp4d, a reversible TG2 inhibitor, did not affect cell death in response to oxygen-glucose deprivation. These findings indicate that the pro-cell death effects of TG2 are dependent on its localization to the cytosol and independent of its transamidation activity. Further, the conformational state of TG2 is likely an important determinant in cell survival and the prominent function of TG2 in ischemic cell death is as a scaffold to modulate cellular processes.

  12. Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

    Science.gov (United States)

    Verma, Amit K; Diwan, Danish; Raut, Sandeep; Dobriyal, Neha; Brown, Rebecca E; Gowda, Vinita; Hines, Justin K; Sahi, Chandan

    2017-06-07

    Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations. Copyright © 2017 Verma et al.

  13. Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA.

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    Sen Li

    Full Text Available Intracellular pH (pHi and Ca(2+ regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+. The sources of the Ca(2+ increase are from the endoplasmic reticulum (ER Ca(2+ pools as well as from Ca(2+ influx. The store-mobilization component of the Ca(2+ increase induced by the pHi rise was not sensitive to antagonists for either IP(3-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+-ATPase (SERCA, leading to depletion of the ER Ca(2+ store. We further showed that the physiological consequence of depletion of the ER Ca(2+ store by pHi rise is the activation of store-operated channels (SOCs of Orai1 and Stim1, leading to increased Ca(2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+ leak from ER pools followed by Ca(2+ influx via SOCs.

  14. Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance

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    Baksh Shairaz

    2007-02-01

    Full Text Available Abstract Background The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC is associated with frequent treatment failures (~90% in metastatic cases, and these tumors frequently contain PTEN abnormalities. Results Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis. Conclusion Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories.

  15. Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance

    Science.gov (United States)

    Lin, Pei-Yin; Fosmire, Susan P; Park, See-Hyoung; Park, Jin-Young; Baksh, Shairaz; Modiano, Jaime F; Weiss, Robert H

    2007-01-01

    Background The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC) is associated with frequent treatment failures (~90% in metastatic cases), and these tumors frequently contain PTEN abnormalities. Results Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis. Conclusion Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories. PMID:17300726

  16. Ultra-small, highly stable, and sensitive dual nanosensors for imaging intracellular oxygen and pH in cytosol.

    Science.gov (United States)

    Wang, Xu-dong; Stolwijk, Judith A; Lang, Thomas; Sperber, Michaela; Meier, Robert J; Wegener, Joachim; Wolfbeis, Otto S

    2012-10-17

    We report on the first dual nanosensors for imaging of pH values and oxygen partial pressure in cells. The sensors have a unique nanostructure in that a soft core structure is rigidized with a silane reagent, while poly(ethylene glycol) chains form an outer shell. Lipophilic oxygen-sensitive probes and reference dyes are encapsulated inside the hydrophobic core, while a pH-sensitive probe is covalently attached to the poly(ethylene glycol) end-group on the shell. The core/shell structure renders the nanosensors well dispersed and highly stable in various kinds of aqueous media. Their average size is 12 nm, and they respond to both pH and oxygen in the physiological range. They do not pass cell membranes, but can be internalized into the cellular cytosol by electroporation, upon which they enable sensing and imaging of pH values and oxygen with high spatial resolution. The nanosensor strategy shown here is expected to be applicable to the development of various other kinds of multiple nanosensors for in vivo studies.

  17. Cytosolic prostaglandin E2 synthase (cPGES) expression is decreased in discrete cortical regions in psychiatric disease.

    Science.gov (United States)

    Maida, Mary E; Hurley, Sean D; Daeschner, Jo Anna; Moore, Amy H; O'Banion, M Kerry

    2006-08-04

    The number of adults in the US affected by bipolar disorder, depression, or schizophrenia is approaching 15 million. Despite decades of research, etiologies of these illnesses remain elusive. Theories of aberrant brain morphology, neurotransmission, and signal conduction have provided the heuristic framework for a large body of literature, with attention focused upon hypotheses of monoamine signaling underlying psychiatric disease. More recently, attention has turned to potential contributions of other signaling pathways, including the arachidonic acid cascade and generation of prostaglandins (PG). To determine the potential involvement of the pathways leading to PGE2 synthesis in psychiatric disease, immunohistochemistry and immunoblotting were performed to measure regional expression of the cyclooxygenases (COX) and one of the terminal PGE2 synthases (PGES) in postmortem tissue provided by The Stanley Medical Research Institute. For normal, bipolar, depressed, and schizophrenic subjects, COX-1 and COX-2 protein levels did not differ across region and patient populations. In contrast, there was a significant effect of diagnosis on cytosolic PGES (cPGES) protein levels in the frontal cortex, with remarkable decreases observed in all psychiatric groups relative to normal tissue (P bipolar subjects. Evaluation of medicated vs. non-medicated subjects revealed a significant effect of medication on cPGES expression in the frontal cortex of bipolar, but not depressed or schizophrenic subjects. These novel findings further support hypotheses of abnormalities in fatty acid and phospholipid metabolism in regions associated with psychiatric disease.

  18. Polysaccharide fraction from higher plants which strongly interacts with the cytosolic phosphorylase isozyme. I. Isolation and characterization. [Spinacia oleracea L. ; Pisum sativum L

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    Yang, Yi; Steup, M. (Botanisches Institut der Westfaelischen Wilhelms-Universitaet Muenster (West Germany))

    1990-11-01

    From leaves of Spinacia oleracea L. or from Pisum sativum L. and from cotyledons of germinating pea seeds a high molecular weight polysaccharide fraction was isolated. The apparent size of the fraction, as determined by gel filtration, was similar to that of dextran blue. Following acid hydrolysis the monomer content of the polysaccharide preparation was studied using high pressure liquid and thin layer chromatography. Glucose, galactose, arabinose, and ribose were the main monosaccharide compounds. The native polysaccharide preparation interacted strongly with the cytosolic isozyme of phosphorylase (EC 2.4.1.1). Interaction with the plastidic phosphorylase isozyme(s) was by far weaker. Interaction with the cytosolic isozyme was demonstrated by affinity electrophoresis, kinetic measurements, and by {sup 14}C-labeling experiments in which the glucosyl transfer from ({sup 14}C)glucose 1-phosphate to the polysaccharide preparation was monitored.

  19. Metabolism by rat liver cytosol of illudin S, a toxic substance of Lampteromyces japonicus. II. Characterization of illudin S-metabolizing enzyme.

    Science.gov (United States)

    Tanaka, K; Inoue, T; Kadota, S; Kikuchi, T

    1992-01-01

    1. Enzyme systems responsible for formation of cyclopropane ring-cleavage metabolites (M1 and M2) of illudin S in rat liver were characterized. 2. The enzymes were localized in the cytosol fraction and utilized NADPH alone as electron donor; they were not affected by oxygen and had low pH optima. 3. Formation of metabolites M1 and M2 was inhibited completely by dicumarol (10(-4) M), an inhibitor of DT-diaphorase. 4. Menadione (10(-4) M) and quercetin (10(-4) M) both inhibited formation of M1 and M2 by 35% and 15%, respectively, but quinacrine, barbital, pyrazole and p-chloromercuribenzoic acid had no significant effect. 5. Results show that the enzyme systems may differ from DT-diaphorase, aldehyde oxidase, xanthine oxidase, ketone reductase, aldose reductase, aldehyde reductase and alcohol dehydrogenase, known cytosolic enzymes responsible for xenobiotic metabolism.

  20. A Novel Cytosolic Isoform of Mitochondrial Trans-2-Enoyl-CoA Reductase Enhances Peroxisome Proliferator-Activated Receptor α Activity

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    Dong-Gyu Kim

    2014-06-01

    Full Text Available BackgroundMitochondrial trans-2-enoyl-CoA reductase (MECR is involved in mitochondrial synthesis of fatty acids and is highly expressed in mitochondria. MECR is also known as nuclear receptor binding factor-1, which was originally reported with yeast two-hybrid screening as a binding protein of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα. However, MECR and PPARα are localized at different compartment, mitochondria, and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for functional interaction between MECR and PPARα.MethodsTo identify the expression pattern of MECR and the cytosolic form of MECR (cMECR, we performed reverse transcription polymerase chain reaction (RT-PCR with various tissue samples from Sprague-Dawley rats. To confirm the interaction between cMECR and PPARα, we performed several binding assays such as yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation. To observe subcellular localization of these proteins, immunocytochemistry was performed. A luciferase assay was used to measure PPARα activity.ResultsWe provide evidence of an alternatively spliced variant of the rat MECR gene that yields cMECR. The cMECR lacks the N-terminal 76 amino acids of MECR and shows uniform distribution in the cytoplasm and nucleus of HeLa cells. cMECR directly bound PPARα in the nucleus and increased PPARα-dependent luciferase activity in HeLa cells.ConclusionWe found the cytosolic form of MECR (cMECR was expressed in the cytosolic and/or nuclear region, directly binds with PPARα, and enhances PPARα activity.

  1. A Novel Cytosolic Isoform of Mitochondrial Trans-2-Enoyl-CoA Reductase Enhances Peroxisome Proliferator-Activated Receptor α Activity.

    Science.gov (United States)

    Kim, Dong-Gyu; Yoo, Jae Cheal; Kim, Eunju; Lee, Young-Sun; Yarishkin, Oleg V; Lee, Da Yong; Lee, Kun Ho; Hong, Seong-Geun; Hwang, Eun Mi; Park, Jae-Yong

    2014-06-01

    Mitochondrial trans-2-enoyl-CoA reductase (MECR) is involved in mitochondrial synthesis of fatty acids and is highly expressed in mitochondria. MECR is also known as nuclear receptor binding factor-1, which was originally reported with yeast two-hybrid screening as a binding protein of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). However, MECR and PPARα are localized at different compartment, mitochondria, and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for functional interaction between MECR and PPARα. To identify the expression pattern of MECR and the cytosolic form of MECR (cMECR), we performed reverse transcription polymerase chain reaction (RT-PCR) with various tissue samples from Sprague-Dawley rats. To confirm the interaction between cMECR and PPARα, we performed several binding assays such as yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation. To observe subcellular localization of these proteins, immunocytochemistry was performed. A luciferase assay was used to measure PPARα activity. We provide evidence of an alternatively spliced variant of the rat MECR gene that yields cMECR. The cMECR lacks the N-terminal 76 amino acids of MECR and shows uniform distribution in the cytoplasm and nucleus of HeLa cells. cMECR directly bound PPARα in the nucleus and increased PPARα-dependent luciferase activity in HeLa cells. We found the cytosolic form of MECR (cMECR) was expressed in the cytosolic and/or nuclear region, directly binds with PPARα, and enhances PPARα activity.

  2. Cytosolic phospholipase A2-α expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours.

    LENUS (Irish Health Repository)

    Caiazza, F

    2011-01-18

    The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)α (cPLA(2)α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)α expression correlated with EGFR\\/HER2 over-expression in a small number of breast cancer cell lines.

  3. Regulation of the translocation of phosphatidate phosphohydrolase between the cytosol and the endoplasmic reticulum of rat liver. Effects of unsaturated fatty acids, spermine, nucleotides, albumin and chlorpromazine.

    OpenAIRE

    Hopewell, R; Martin-Sanz, P; Martin, A; Saxton, J; Brindley, D N

    1985-01-01

    The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, alpha-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c ...

  4. The iron-responsive element (IRE)/iron-regulatory protein 1 (IRP1)–cytosolic aconitase iron-regulatory switch does not operate in plants

    Science.gov (United States)

    Arnaud, Nicolas; Ravet, Karl; Borlotti, Andrea; Touraine, Brigitte; Boucherez, Jossia; Fizames, Cécile; Briat, Jean-François; Cellier, Françoise; Gaymard, Frédéric

    2007-01-01

    Animal cytosolic ACO (aconitase) and bacteria ACO are able to switch to RNA-binding proteins [IRPs (iron-regulatory proteins)], thereby playing a key role in the regulation of iron homoeostasis. In the model plant Arabidopsis thaliana, we have identified three IRP1 homologues, named ACO1–3. To determine whether or not they may encode functional IRP proteins and regulate iron homoeostasis in plants, we have isolated loss-of-function mutants in the three genes. The aco1-1 and aco3-1 mutants show a clear decrease in cytosolic ACO activity. However, none of the mutants is affected in respect of the accumulation of the ferritin transcript or protein in response to iron excess. cis-acting elements potentially able to bind to the IRP have been searched for in silico in the Arabidopsis genome. They appear to be very rare sequences, found in the 5′-UTR (5′-untranslated region) or 3′-UTR of a few genes unrelated to iron metabolism. They are therefore unlikely to play a functional role in the regulation of iron homoeostasis. Taken together, our results demonstrate that, in plants, the cytosolic ACO is not converted into an IRP and does not regulate iron homoeostasis. In contrast with animals, the RNA binding activity of plant ACO, if any, would be more likely to be attributable to a structural element, rather than to a canonical sequence. PMID:17437406

  5. Profiling of cytosolic and mitochondrial H2O2production using the H2O2-sensitive protein HyPer in LPS-induced microglia cells.

    Science.gov (United States)

    Park, Junghyung; Lee, Seunghoon; Lee, Hyun-Shik; Lee, Sang-Rae; Lee, Dong-Seok

    2017-07-27

    Dysregulation of the production of pro-inflammatory mediators in microglia exacerbates the pathologic process of neurodegenerative disease. ROS actively affect microglia activation by regulating transcription factors that control the expression of pro-inflammatory genes. However, accurate information regarding the function of ROS in different subcellular organelles has not yet been established. Here, we analyzed the pattern of cytosolic and mitochondrial H 2 O 2 formation in LPS-activated BV-2 microglia using the H 2 O 2- sensitive protein HyPer targeted to specific subcellular compartments. Our results show that from an early time, cytosolic H 2 O 2 started increasing constantly, whereas mitochondrial H 2 O 2 rapidly increased later. In addition, we found that MAPK affected cytosolic H 2 O 2 , but not mitochondrial H 2 O 2 . Consequently, our study provides the basic information about subcellular H 2 O 2 generation in activated microglia, and a useful tool for investigating molecular targets that can modulate neuroinflammatory responses. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Glutathione monoethyl ester and inhibition of the oxyhemoglobin-induced increase in cytosolic calcium in cultured smooth-muscle cells.

    Science.gov (United States)

    Arai, T; Takeyama, N; Tanaka, T

    1999-03-01

    The mechanism of arterial vasoconstriction caused by oxyhemoglobin production after subarachnoid hemorrhage was investigated. Using a fluorescent Ca++ indicator (fura-2 acetoxymethyl ester), the change in the cytosolic intracellular Ca++ concentration, [Ca++]i. was measured in cultured rat vascular smooth-muscle cells exposed to oxyhemoglobin and other substances. Oxyhemoglobin induced transient elevation of smooth-muscle cell [Ca++]i in either the presence or absence of ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid, indicating that Ca++ released by oxyhemoglobin was derived from [Ca++]i stores. In contrast, methemoglobin had no effect on the smooth-muscle cells. Exposure of the cells to reactive oxygen species generated by xanthine plus xanthine oxidase yielded the same results as with oxyhemoglobin, that is, transient elevation of smooth-muscle cell [Ca++]i. Procaine (a Ca++ channel blocker) failed to inhibit the oxyhemoglobin-induced elevation of [Ca++]i. Ryanodine (a Ca++ channel opener) plus oxyhemoglobin caused markedly greater elevation of [Ca++]i than ryanodine alone, whereas thapsigargin (an adenosine triphosphate [ATP]-dependent Ca++ pump inhibitor) plus oxyhemoglobin had no additional effect when compared with thapsigargin alone. The oxyhemoglobin-induced elevation of [Ca++]i could be blocked by an Fe++ chelator (ferene), but not by an Fe chelator (deferoxamine mesylate). Treatment with either dithiothreitol or glutathione monoethyl ester markedly inhibited the oxyhemoglobin-induced elevation of [Ca++]i. These results indicate that Fe++-catalyzed hydroxyl radicals generated from oxyhemoglobin-derived free radicals induce the elevation of [Ca++]i by inhibiting the ATP-dependent Ca++ pump rather than the Ca++ channels in the sarcoplasmic reticulum and that thiols may prevent Ca++ pump inactivation by inhibiting the oxidation of membrane sulfhydryl groups.

  7. Targeting the cytosolic innate immune receptors RIG-I and MDA5 effectively counteracts cancer cell heterogeneity in glioblastoma.

    Science.gov (United States)

    Glas, Martin; Coch, Christoph; Trageser, Daniel; Dassler, Juliane; Simon, Matthias; Koch, Philipp; Mertens, Jerome; Quandel, Tamara; Gorris, Raphaela; Reinartz, Roman; Wieland, Anja; Von Lehe, Marec; Pusch, Annette; Roy, Kristin; Schlee, Martin; Neumann, Harald; Fimmers, Rolf; Herrlinger, Ulrich; Brüstle, Oliver; Hartmann, Gunther; Besch, Robert; Scheffler, Björn

    2013-06-01

    Cellular heterogeneity, for example, the intratumoral coexistence of cancer cells with and without stem cell characteristics, represents a potential root of therapeutic resistance and a significant challenge for modern drug development in glioblastoma (GBM). We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells. We used short-term expanded patient-specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Specifically, we analyzed cells from the tumor core versus "residual GBM cells" derived from the tumor resection margin as well as stem cell-enriched primary cultures versus specimens without stem cell properties. A portfolio of human, nontumor neural cells was used as a control for these studies. The expression of RIG-I and MDA5 could be induced in all of these cells. Receptor stimulation with their respective ligands, p(I:C) and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system. Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity. By contrast, p(I:C) and 3pRNA induced only little toxicity in human nonmalignant neural cells. Granted that the challenge of effective central nervous system (CNS) delivery can be overcome, targeting of RIG-I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain. Copyright © 2013 AlphaMed Press.

  8. Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells.

    Science.gov (United States)

    Yao, Mu; Xie, Chanlu; Kiang, Mei-Yee; Teng, Ying; Harman, David; Tiffen, Jessamy; Wang, Qian; Sved, Paul; Bao, Shisan; Witting, Paul; Holst, Jeff; Dong, Qihan

    2015-10-27

    Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A2α (cPLA2α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA2α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA2α with a phosphorylation at Ser505 was lower compared to their proliferating state. Conversely, the phospho-cPLA2α levels were resurgent during the induction of cell cycle re-entry. Pharmacological inhibition of cPLA2α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G0/G1 and low percentage of cells in S and G2/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA2α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phospho-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA2α plays an important role in cell cycle re-entry by quiescent prostate cancer cells.

  9. P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships

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    Di Pietro A.

    1999-01-01

    Full Text Available Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp, a plasma membrane ATP-binding cassette (ABC transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR. In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

  10. The cytosolic tail of the Golgi apyrase Ynd1 mediates E4orf4-induced toxicity in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Karin Mittelman

    Full Text Available The adenovirus E4 open reading frame 4 (E4orf4 protein contributes to regulation of the progression of virus infection. When expressed individually, E4orf4 was shown to induce non-classical transformed cell-specific apoptosis in mammalian cells. At least some of the mechanisms underlying E4orf4-induced toxicity are conserved from yeast to mammals, including the requirement for an interaction of E4orf4 with protein phosphatase 2A (PP2A. A genetic screen in yeast revealed that the Golgi apyrase Ynd1 associates with E4orf4 and contributes to E4orf4-induced toxicity, independently of Ynd1 apyrase activity. Ynd1 and PP2A were shown to contribute additively to E4orf4-induced toxicity in yeast, and to interact genetically and physically. A mammalian orthologue of Ynd1 was shown to bind E4orf4 in mammalian cells, confirming the evolutionary conservation of this interaction. Here, we use mutation analysis to identify the cytosolic tail of Ynd1 as the protein domain required for mediation of the E4orf4 toxic signal and for the interaction with E4orf4. We also show that E4orf4 associates with cellular membranes in yeast and is localized at their cytoplasmic face. However, E4orf4 is membrane-associated even in the absence of Ynd1, suggesting that additional membrane proteins may mediate E4orf4 localization. Based on our results and on a previous report describing a collection of Ynd1 protein partners, we propose that the Ynd1 cytoplasmic tail acts as a scaffold, interacting with a multi-protein complex, whose targeting by E4orf4 leads to cell death.

  11. PGC-1{alpha} accelerates cytosolic Ca{sup 2+} clearance without disturbing Ca{sup 2+} homeostasis in cardiac myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Min, E-mail: chenminyx@gmail.com [Institute of Molecular Medicine, State Key Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871 (China); Yunnan Centers for Diseases Prevention and Control, Kunming 650022 (China); Wang, Yanru [Institute of Molecular Medicine, State Key Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871 (China); Qu, Aijuan [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2010-06-11

    Energy metabolism and Ca{sup 2+} handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1{alpha}) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1{alpha} in cardiac Ca{sup 2+} signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1{alpha} via adenoviral transduction. Our data shows that overexpressing PGC-1{alpha} improved myocyte contractility without increasing the amplitude of Ca{sup 2+} transients, suggesting that myofilament sensitivity to Ca{sup 2+} increased. Interestingly, the decay kinetics of global Ca{sup 2+} transients and Ca{sup 2+} waves accelerated in PGC-1{alpha}-expressing cells, but the decay rate of caffeine-elicited Ca{sup 2+} transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca{sup 2+}-ATPase (SERCA2a), but not Na{sup +}/Ca{sup 2+} exchange (NCX) contribute to PGC-1{alpha}-induced cytosolic Ca{sup 2+} clearance. Furthermore, PGC-1{alpha} induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1{alpha} did not disturb cardiac Ca{sup 2+} homeostasis, because SR Ca{sup 2+} load and the propensity for Ca{sup 2+} waves remained unchanged. These data suggest that PGC-1{alpha} can ameliorate cardiac Ca{sup 2+} cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1{alpha}-calcium handing pathway sheds new light on the role of PGC-1{alpha} in the therapy of cardiac diseases.

  12. Cytosolic phospholipase A2 alpha/arachidonic acid signaling mediates depolarization-induced suppression of excitation in the cerebellum.

    Directory of Open Access Journals (Sweden)

    De-Juan Wang

    Full Text Available Depolarization-induced suppression of excitation (DSE at parallel fiber-Purkinje cell synapse is an endocannabinoid-mediated short-term retrograde plasticity. Intracellular Ca(2+ elevation is critical for the endocannabinoid production and DSE. Nevertheless, how elevated Ca(2+ leads to DSE is unclear.We utilized cytosolic phospholipase A(2 alpha (cPLA(2α knock-out mice and whole-cell patch clamp in cerebellar slices to observed the action of cPLA(2α/arachidonic acid signaling on DSE at parallel fiber-Purkinje cell synapse. Our data showed that DSE was significantly inhibited in cPLA(2α knock-out mice, which was rescued by arachidonic acid. The degradation enzyme of 2-arachidonoylglycerol (2-AG, monoacylglycerol lipase (MAGL, blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine (AEA, fatty acid amide hydrolase (FAAH, did not affect DSE. These results suggested that 2-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K(+, indicating that large conductance Ca(2+-activated potassium channel (BK is sufficient to inhibit cPLA(2α/arachidonic acid-mediated DSE. In addition, we showed that the release of 2-AG was independent of soluble NSF attachment protein receptor (SNARE, protein kinase C and protein kinase A.Our data first showed that cPLA(2α/arachidonic acid/2-AG signaling pathway mediates DSE at parallel fiber-Purkinje cell synapse.

  13. Specific interactions of steroids, arylhydrocarbons and flavonoids with progesterone receptors from the cytosol of the fungus Rhizopus nigricans.

    Science.gov (United States)

    Lenasi, Helena; Breskvar, Katja

    2004-08-01

    Rhizopus nigricans (R. nigricans) transforms fungitoxic progesterone into the less toxic 11alpha-hydroxyprogesterone which is then able to exit the mycelia into the surrounding water. Hydroxylation of progesterone is an inducible process in which cytosolic progesterone receptors could be involved. In the present study, we characterised receptors with respect to ligand specificity and to their involvement in progesterone induction of hydroxylase. EC(50) values of different ligands (steroids, xenobiotic arylhydrocarbons and natural flavonoids) were determined by competition studies using 40nM ((3)H)progesterone. C21 and C19 3-oxo-4-ene steroids were good competitors (EC(50) of progesterone 2.3 +/- 0.1 x 10(-7)M, EC(50) of androsten-3,17-dione 24 +/- 2 x 10(-7)M). The presence of hydroxyl groups in steroids significantly decreased the affinity for receptors. The arylhydrocarbons alpha-naphthoflavone and ketoconazole exhibited EC(50) values of 0.3 +/- 0.01 x 10(-7)M and 27 +/- 5 x 10(-7)M, respectively, whereas beta-naphthoflavone and benzo(a)pyrene were not able to displace labelled progesterone completely. The competition curves obtained by natural flavonoids also did not reach the bottom level of non-labelled progesterone, indicating the interaction at some allosteric binding site(s) of progesterone receptors. All ligands were examined for their involvement in progesterone-hydroxylase induction. Steroid agonists induced the enzyme in a dose-dependent manner in accordance with their affinity for receptors, whereas arylhydrocarbons and natural flavonoids did not induce the enzyme. The agonistic action of steroids, together with the antagonistic action of alpha-naphthoflavone, strongly suggests the involvement of progesterone receptors in progesterone signalling resulting in the induction of progesterone-hydroxylase.

  14. Expression analysis of cytosolic DNA-sensing pathway genes in the intestinal mucosal layer of necrotic enteritis-induced chicken.

    Science.gov (United States)

    Rengaraj, Deivendran; Truong, Anh Duc; Lee, Sung-Hyen; Lillehoj, Hyun S; Hong, Yeong Ho

    2016-02-01

    Necrotic enteritis (NE) is a serious problem to the poultry farms, which report NE outbreaks more than once per year, as a result of the inappropriate use of antibiotics in the feed. The NE affected bird die rapidly as a result of various pathophysiological complications in the intestine and immune system. Also, several studies have reported that the genes exclusively related to intestine and immune functions are significantly altered in response to NE. In this study, NE was induced in two genetically disparate chicken lines that are resistant (line 6.3) and sensitive (line 7.2) to avian leukosis and Marek's disease. The intestinal mucosal layer was collected from NE-induced and control chickens, and subjected to RNA-sequencing analysis. The involvement of differentially expressed genes in the intestinal mucosal layer of line 6.3 and 7.2 with the immune system-related pathways was investigated. Among the identified immune system-related pathways, a candidate pathway known as chicken cytosolic DNA-sensing pathway (CDS pathway) was selected for further investigation. RNA-sequencing and pathway analysis identified a total of 21 genes that were involved in CDS pathway and differentially expressed in the intestinal mucosal layer of lines 6.3 and 7.2. The expression of CDS pathway genes was further confirmed by real-time qPCR. In the results, a majority of the CDS pathway genes were significantly altered in the NE-induced intestinal mucosal layer from lines 6.3 and 7.2. In conclusion, our study indicate that NE seriously affects several genes involved in innate immune defense and foreign DNA sensing mechanisms in the chicken intestinal mucosal layer. Identifying the immune genes affected by NE could be an important evidence for the protective immune response to NE-causative pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Proteomic comparison of the cytosolic proteins of three Bifidobacterium longum human isolates and B. longum NCC2705

    Directory of Open Access Journals (Sweden)

    Champomier-Vergès Marie-Christine

    2010-01-01

    Full Text Available Abstract Background Bifidobacteria are natural inhabitants of the human gastrointestinal tract. In full-term newborns, these bacteria are acquired from the mother during delivery and rapidly become the predominant organisms in the intestinal microbiota. Bifidobacteria contribute to the establishment of healthy intestinal ecology and can confer health benefits to their host. Consequently, there is growing interest in bifidobacteria, and various strains are currently used as probiotic components in functional food products. However, the probiotic effects have been reported to be strain-specific. There is thus a need to better understand the determinants of the observed benefits provided by these probiotics. Our objective was to compare three human B. longum isolates with the sequenced model strain B. longum NCC2705 at the chromosome and proteome levels. Results Pulsed field electrophoresis genotyping revealed genetic heterogeneity with low intraspecies strain relatedness among the four strains tested. Using two-dimensional gel electrophoresis, we analyzed qualitative differences in the cytosolic protein patterns. There were 45 spots that were present in some strains and absent in others. Spots were excised from the gels and subjected to peptide mass fingerprint analysis for identification. The 45 spots represented 37 proteins, most of which were involved in carbohydrate metabolism and cell wall or cell membrane synthesis. Notably, the protein patterns were correlated with differences in cell membrane properties like surface hydrophobicity and cell agglutination. Conclusion These results showed that proteomic analysis can be valuable for investigating differences in bifidobacterial species and may provide a better understanding of the diversity of bifidobacteria and their potential use as probiotics.

  16. Cytosolic phospholipase A2 activation correlates with HER2 overexpression and mediates estrogen-dependent breast cancer cell growth.

    LENUS (Irish Health Repository)

    Caiazza, Francesco

    2010-05-01

    Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) catalyzes the hydrolysis of membrane glycerol-phospholipids to release arachidonic acid as the first step of the eicosanoid signaling pathway. This pathway contributes to proliferation in breast cancer, and numerous studies have demonstrated a crucial role of cyclooxygenase 2 and prostaglandin E(2) release in breast cancer progression. The role of cPLA(2)alpha activation is less clear, and we recently showed that 17beta-estradiol (E2) can rapidly activate cPLA(2)alpha in MCF-7 breast cancer cells. Overexpression or gene amplification of HER2 is found in approximately 30% of breast cancer patients and correlates with a poor clinical outcome and resistance to endocrine therapy. This study reports the first evidence for a correlation between cPLA(2)alpha enzymatic activity and overexpression of the HER2 receptor. The activation of cPLA(2)alpha in response to E2 treatment was biphasic with the first phase dependent on trans-activation through the matrix metalloproteinase-dependent release of heparin-bound epidermal growth factor. EGFR\\/HER2 heterodimerization resulted in downstream signaling through the ERK1\\/2 cascade to promote cPLA(2)alpha phosphorylation at Ser505. There was a correlation between HER2 and cPLA(2)alpha expression in six breast cancer cell lines examined, and inhibition of HER2 activation or expression in the SKBR3 cell line using herceptin or HER2-specific small interfering RNA, respectively, resulted in decreased activation and expression of cPLA(2)alpha. Pharmacological blockade of cPLA(2)alpha using a specific antagonist suppressed the growth of both MCF-7 and SKBR3 cells by reducing E2-induced proliferation and by stimulating cellular apoptosis and necrosis. This study highlights cPLAalpha(2) as a potential target for therapeutic intervention in endocrine-dependent and endocrine-independent breast cancer.

  17. H2O2augments cytosolic calcium in nucleus tractus solitarii neurons via multiple voltage-gated calcium channels.

    Science.gov (United States)

    Ostrowski, Tim D; Dantzler, Heather A; Polo-Parada, Luis; Kline, David D

    2017-05-01

    Reactive oxygen species (ROS) play a profound role in cardiorespiratory function under normal physiological conditions and disease states. ROS can influence neuronal activity by altering various ion channels and transporters. Within the nucleus tractus solitarii (nTS), a vital brainstem area for cardiorespiratory control, hydrogen peroxide (H 2 O 2 ) induces sustained hyperexcitability following an initial depression of neuronal activity. The mechanism(s) associated with the delayed hyperexcitability are unknown. Here we evaluate the effect(s) of H 2 O 2 on cytosolic Ca 2+ (via fura-2 imaging) and voltage-dependent calcium currents in dissociated rat nTS neurons. H 2 O 2 perfusion (200 µM; 1 min) induced a delayed, slow, and moderate increase (~27%) in intracellular Ca 2+ concentration ([Ca 2+ ] i ). The H 2 O 2 -mediated increase in [Ca 2+ ] i prevailed during thapsigargin, excluding the endoplasmic reticulum as a Ca 2+ source. The effect, however, was abolished by removal of extracellular Ca 2+ or the addition of cadmium to the bath solution, suggesting voltage-gated Ca 2+ channels (VGCCs) as targets for H 2 O 2 modulation. Recording of the total voltage-dependent Ca 2+ current confirmed H 2 O 2 enhanced Ca 2+ entry. Blocking VGCC L, N, and P/Q subtypes decreased the number of cells and their calcium currents that respond to H 2 O 2 The number of responder cells to H 2 O 2 also decreased in the presence of dithiothreitol, suggesting the actions of H 2 O 2 were dependent on sulfhydryl oxidation. In summary, here, we have shown that H 2 O 2 increases [Ca 2+ ] i and its Ca 2+ currents, which is dependent on multiple VGCCs likely by oxidation of sulfhydryl groups. These processes presumably contribute to the previously observed delayed hyperexcitability of nTS neurons in in vitro brainstem slices. Copyright © 2017 the American Physiological Society.

  18. Cytosolic calcium elevation induced by orexin/hypocretin in granule cell domain cells of the rat cochlear nucleus in vitro.

    Science.gov (United States)

    Nakamura, Yuki; Miura, Shinya; Yoshida, Takashi; Kim, Juhyon; Sasaki, Kazuo

    2010-08-01

    Using rat brain slice preparations, we examined the effect of orexin on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) in the granule cell domain (GCD) cells of the cochlear nucleus that carry non-auditory information to the dorsal cochlear nucleus. Application of orexin concentration-dependently increased [Ca(2+)](i), and in two thirds of GCD cells these increases persisted in the presence of tetrodotoxin. There was no significant difference between the dose-response curve for orexin-A and that for orexin-B. Extracellular Ca(2+) removal abolished the [Ca(2+)](i) elevation induced by orexin-B, whereas depletion of intracellular Ca(2+) stores had no effect. The orexin-B-induced elevation of [Ca(2+)](i) was not blocked by inhibitors of reverse-mode Na(+)/Ca(2+) exchanger (NCX) and nonselective cation channel, whereas it was blocked by lowering the extracellular Na(+) or by applying inhibitors of forward-mode NCX and voltage-gated R- and T-type Ca(2+) channels. The ORX-B-induced increase in [Ca(2+)](i) was also blocked by inhibitors of adenylcyclase (AC) and protein kinase A (PKA), but not by inhibitors of phosphatidylcholine-specific and phosphatidylinositol-specific phospholipase C. In electrophysiological experiments using whole-cell patch clamp recordings, half of GCD cells were depolarized by orexin-B, and the depolarization was abolished by a forward-mode NCX inhibitor. These results suggest that orexin increases [Ca(2+)](i) postsynaptically via orexin 2 receptors, and the increase in [Ca(2+)](i) is induced via the AC-PKA-forward-mode NCX-membrane depolarization-mediated activation of voltage-gated R- and T-type Ca(2+) channels. The results further support the hypothesis that the orexin system participates in integrating neural systems that are involved in arousal, sensory processing, energy homeostasis and autonomic function. Copyright 2010 Elsevier Inc. All rights reserved.

  19. Computational Exploration of Natural Compounds to Target Cytosolic Phospholipase A 2 Protein: A Novel Therapeutic Target for Spinal Cord Injury.

    Science.gov (United States)

    Fan, Hongwu; Wang, Shengqun; Zhao, Qiheng; Qin, Zhigang

    2018-01-22

    Cytosolic phospholipase A2 (cPLA2), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of spinal cord injury (SCI). The expression and activation of Cpla2is significantly higher in SCI, leading to neuronal death in spinal cord tissue. Novel strategies are needed to substantially reverse the effect of cPLA2 activation; one such strategy is inhibiting cPLA2 by jamming its lipid binding C2 domain. To develop a much needed strategy to treat SCI we used a computer aided drug design (CADD) method to discover novel cPLA2 inhibitors. we used a natural chemiome database for virtual screening, from which we selected the compounds exhibiting the greatest drug-likeliness properties for molecular docking simulation analysis. We studied the interaction of lead compounds at the atomic level; the results yielded a cPLA2 inhibitor of natural origin with the potential for ameliorating secondary tissue damage and promoting recovery of function after SCI. The top compound, lead 4exibited a binding energy of -10.02 Kcal/mol and formed three hydrogen bonds with the lipid binding C2 domain of the cPLA2 protein. An evaluation of cell cytotoxicity revealed an IC50 for lead4 of 134.2 ± 6.8 µM. An in-vitro analysis of lead4 is indicated anti-apoptotic activity via a decrease in caspase-3 expression. We used the CADD method to make a novel lead discovery for the treatment of SCIusing compounds of natural origin. The selected natural compounds are non-toxic promising drugs against cPLA2 protein, allowing us to limits our focus on single compound for future in-vitro and in-vivo testing. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Palladacycle (BPC) antitumour activity against resistant and metastatic cell lines: the relationship with cytosolic calcium mobilisation and cathepsin B activity.

    Science.gov (United States)

    Bechara, Alexandre; Barbosa, Christiano M V; Paredes-Gamero, Edgar J; Garcia, Daniel M; Silva, Luís S; Matsuo, Alisson L; Nascimento, Fábio D; Rodrigues, Elaine G; Caires, Antonio C F; Smaili, Soraya S; Bincoletto, Claudia

    2014-05-22

    The search for new compounds that induce p53-independent apoptosis is the focus of many studies in cancer biology because these compounds could be more specific and would overcome chemotherapy resistance. In this study, we evaluated the in vitro antitumour activity of a Biphosphinic Palladacycle Complex (BPC) and extended preclinical studies to an in vivo model. Saos-2 cells, a p53-null human osteosarcoma drug-resistant cell line, were treated with BPC in the presence or absence of a cathepsin B inhibitor and a calcium chelator (CA074 and BAPTA-AM, respectively), and several parameters related to apoptosis were evaluated. Preclinical studies were performed with mice that were intravenously inoculated with murine melanoma B16F10-Nex2 cells and treated intraperitoneally (i.p.) with BPC (8 mg/kg/day) for ten consecutive days, when lung metastatic nodules were counted. In vitro data show that BPC induces cell death in Saos-2 cells mainly by apoptosis, which was accompanied by the effector caspase-3 activation. These events are most likely related to Bax translocation and increased cytosolic calcium mobilisation, mainly from intracellular compartments. Lysosomal Membrane Permeabilisation (LMP) was also observed after 12 h of BPC exposure. Interestingly, BAPTA-AM and CA074 significantly decreased BPC cytotoxicity, suggesting that both calcium and cathepsin B are required for BPC antitumour activity. In vivo studies demonstrated that BPC protects mice against murine metastatic melanoma. In conclusion, BPC complex is an effective anticancer compound against metastatic murine melanoma. This complex is cytotoxic to the drug-resistant osteosarcoma Saos-2 human tumour cells by inducing apoptosis triggered by calcium signalling and a lysosomal-dependent pathway. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. The Tumor Cytosol miRNAs, Fluid miRNAs and Exosome miRNAs in Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xin eQin

    2015-01-01

    Full Text Available The focus of this review is to provide an update on the progress of microRNAs (miRNAs as potential biomarkers for lung cancer. miRNAs are single-stranded, small noncoding RNAs that regulate gene expression and show tissue-specific signatures. Accumulating evidence indicates that miRNA expression patterns represent the in vivo status in physiology and disease. Moreover, miRNAs are stable in serum and other clinically convenient and available tissue sources, so they are being developed as biomarkers for cancer and other diseases. Cancer is currently the primary driver of the field, but miRNA biomarkers are being developed for many other diseases such as cardiovascular and central nervous system diseases. Here we examine the framework and scope of the miRNA landscape as it specifically relates to the translation of miRNA expression patterns/signatures into biomarkers for developing diagnostics for lung cancer. We focus on examining tumor cytosol miRNAs, fluid miRNAs, and exosome miRNAs in lung cancer, the connections among these miRNAs, and the potential of miRNA biomarkers for the development of diagnostics. In lung cancer, miRNAs have been studied in both cell populations and in the circulation. However, a major challenge is to develop biomarkers to monitor cancer development and to identify circulating miRNAs that are linked to cancer stage. Importantly, the fact that miRNAs can be successfully harvested from biological fluids allows for the development of biofluid biopsies, in which miRNAs as circulating biomarkers can be captured and analyzed ex vivo. Our hope is that these minimally invasive entities provide a window to the in vivo milieu of the patients without the need for costly, complex invasive procedures, rapidly moving miRNAs from research to the clinic.

  2. Disease specificity of autoantibodies to cytosolic 5'-nucleotidase 1A in sporadic inclusion body myositis versus known autoimmune diseases.

    Science.gov (United States)

    Herbert, Megan K; Stammen-Vogelzangs, Judith; Verbeek, Marcel M; Rietveld, Anke; Lundberg, Ingrid E; Chinoy, Hector; Lamb, Janine A; Cooper, Robert G; Roberts, Mark; Badrising, Umesh A; De Bleecker, Jan L; Machado, Pedro M; Hanna, Michael G; Plestilova, Lenka; Vencovsky, Jiri; van Engelen, Baziel G; Pruijn, Ger J M

    2016-04-01

    The diagnosis of inclusion body myositis (IBM) can be challenging as it can be difficult to clinically distinguish from other forms of myositis, particularly polymyositis (PM). Recent studies have shown frequent presence of autoantibodies directed against cytosolic 5'-nucleotidase 1A (cN-1A) in patients with IBM. We therefore, examined the autoantigenicity and disease specificity of major epitopes of cN-1A in patients with sporadic IBM compared with healthy and disease controls. Serum samples obtained from patients with IBM (n=238), PM and dermatomyositis (DM) (n=185), other autoimmune diseases (n=246), other neuromuscular diseases (n=93) and healthy controls (n=35) were analysed for the presence of autoantibodies using immunodominant cN-1A peptide ELISAs. Autoantibodies directed against major epitopes of cN-1A were frequent in patients with IBM (37%) but not in PM, DM or non-autoimmune neuromuscular diseases (autoimmune diseases, particularly Sjögren's syndrome (SjS; 36%) and systemic lupus erythematosus (SLE; 20%). In summary, we found frequent anti-cN-1A autoantibodies in sera from patients with IBM. Heterogeneity in reactivity with the three immunodominant epitopes indicates that serological assays should not be limited to a distinct epitope region. The similar reactivities observed for SjS and SLE demonstrate the need to further investigate whether distinct IBM-specific epitopes exist. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  3. Bradykinin-evoked changes in cytosolic calcium and membrane currents in cultured bovine pulmonary artery endothelial cells.

    Science.gov (United States)

    Cannell, M B; Sage, S O

    1989-12-01

    1. Cultured bovine pulmonary artery endothelial cells were voltage clamped using a single microelectrode while cytosolic free calcium concentration ([Ca2+]i) was simultaneously measured using the fluorescent calcium indicator, Indo-1. 2. The resting current-voltage relationship was non-linear and exhibited marked inward rectification near the resting potential. In about 60% of cells examined, superfusion of saline resulted in a hyperpolarization and decrease in inward current. This result did not depend on the presence of agonist and is consistent with the presence of extracellular potassium accumulation in restricted spaces around the cell and the known dependence of the inward rectifier. In other cells there was no effect. 3. Resting [Ca2+]i was sensitive to membrane potential, decreasing continuously with membrane depolarization over the range -70 to +60 mV. This result is consistent with a simple pump-leak model and suggests that voltage-dependent calcium channels are not present in these cells. 4. Bradykinin (10 microM) increased [Ca2+]i after a delay of approximately 3 s. [Ca2+]i reached a peak after a further 3 s and declined over several minutes. 5. During the rise in [Ca2+]i evoked by application of bradykinin, there were no changes in the current-voltage relationship of the cell. These results question the role of a receptor-operated non-selective cation channel in mediating the increase in [Ca2+]i. This observation, coupled with the observed delay in the agonist-evoked response suggests that a second messenger system is involved in mediating the increase in [Ca2+]i. 6. Changes in the current-voltage relation started to occur about 30 s after the application of agonist. These changes could be explained by the activation of large-conductance potassium and non-selective cation channels with a reversal potential near 0 mV. The latter channels may mediate the plateau phase of the agonist-evoked response. 7. The results are discussed with respect to the

  4. Cytosolic free calcium dynamics as related to hyphal and colony growth in the filamentous fungal pathogen Colletotrichum graminicola.

    Science.gov (United States)

    Lange, Mario; Peiter, Edgar

    2016-06-01

    Tip growth of pollen tubes and root hairs of plants is oscillatory and orchestrated by tip-focussed variations of cytosolic free calcium ([Ca(2+)]cyt). Hyphae of filamentous fungi are also tubular tip-growing cells, and components of the Ca(2+) signalling machinery, such as Ca(2+) channels and Ca(2+) sensors, are known to be important for fungal growth. In this study, we addressed the questions if tip-focussed [Ca(2+)]cyt transients govern hyphal and whole-colony growth in the maize pathogen Colletotrichum graminicola, and whether colony-wide [Ca(2+)]cyt dynamics rely on external Ca(2+) or internal Ca(2+) stores. Ratiometric fluorescence microscopy of individual hyphae expressing the Ca(2+) reporter Yellow Cameleon 3.6 revealed that Ca(2+) spikes in hyphal tips precede the re-initiation of growth after wounding. Tip-focussed [Ca(2+)]cyt spikes were also observed in undisturbed growing hyphae. They occurred not regularly and at a higher rate in hyphae growing at a medium-glass interface than in those growing on an agar surface. Hyphal tip growth was non-pulsatile, and growth speed was not correlated with the rate of spike occurrence. A possible relationship of [Ca(2+)]cyt spike generation and growth of whole colonies was assessed by using a codon-optimized version of the luminescent Ca(2+) reporter Aequorin. Depletion of extracellular free Ca(2+) abolished [Ca(2+)]cyt spikes nearly completely, but had only a modest effect on colony growth. In a pharmacological survey, some inhibitors targeting Ca(2+) influx or release from internal stores repressed growth strongly. However, although some of those inhibitors also affected [Ca(2+)]cyt spike generation, the effects on both parameters were not correlated. Collectively, the results indicate that tip growth of C. graminicola is non-pulsatile and not mechanistically linked to tip-focused or global [Ca(2+)]cyt spikes, which are likely a response to micro-environmental parameters, such as the physical properties of the

  5. Immune responses of prophenoloxidase and cytosolic manganese superoxide dismutase in the freshwater crayfish Cherax quadricarinatus against a virus and bacterium.

    Science.gov (United States)

    Liu, Yan-Ting; Chang, Chin-I; Hseu, Jinn-Rong; Liu, Kuan-Fu; Tsai, Jyh-Ming

    2013-11-01

    Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cytMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of the above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus. A phylogenetic analysis of the amino acid sequences showed that C. quadricarinatus proPO and cytMnSOD were more closely related to the proPO and cytMnSOD of other crayfish than to those of penaeids, crabs, lobsters, or freshwater prawns. A tissue distribution analysis revealed that proPO was primarily expressed in hemocytes, gills, and the heart, while cytMnSOD was detected in all tissues examined. All of the crayfish artificially infected with white spot syndrome virus (WSSV) died within 4 days. According to a non-lethal dose, there was no mortality in crayfish when infected deliberately with Aeromonas hydrophila. Total hemocyte counts (THCs) had significantly decreased in crayfish at 48 and 72 h after infection with WSSV compared to the control group. In contrast, THCs of crayfish after A. hydrophila challenge had recovered by 48 and 72 h from a lower level at 24 h. There were similar responses in enzyme activities toward WSSV and A. hydrophila infection. Phenoloxidase (PO) and superoxide dismutase (SOD) activities per hemocyte significantly increased from 48 to 72 h compared to the control group. After WSSV challenge, expressions of proPO and cytMnSOD transcripts in hemocytes significantly decreased at 12h, then had respectively recovered and increased at 24 h. At 48-72 h, transcript levels were finally downregulated. No significant differences in the expression profiles of proPO and cytMnSOD were observed between the A. hydrophila-infected and control groups, besides the significant upregulation at 24h post-infection. These results implicate proPO and cytMnSOD in the immune response, and they presented similar expression patterns, although different defense mechanisms may exist for crayfish induced by WSSV and A

  6. Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.

    Science.gov (United States)

    Theler, J M; Mollard, P; Guérineau, N; Vacher, P; Pralong, W F; Schlegel, W; Wollheim, C B

    1992-09-05

    In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still

  7. Cytosolic glutamine synthetase isozymes play redundant roles in ammonium assimilation under low-ammonium conditions in roots of Arabidopsis thaliana.

    Science.gov (United States)

    Konishi, Noriyuki; Saito, Masahide; Imagawa, Fumi; Kanno, Keiich; Yamaya, Tomoyuki; Kojima, Soichi

    2018-01-24

    Ammonium is a major nitrogen source for plants; it is assimilated into glutamine via a reaction catalyzed by glutamine synthetase (GLN). Arabidopsis expresses four cytosolic GLN genes, GLN1;1, GLN1;2, GLN1;3, and GLN1;4, in roots. However, the function and organization of these GLN1 isozymes in ammonium assimilation in roots remain unclear. In this study, we aimed to characterize the four GLN1 isozymes. The levels of growth of wild type and gln1 single- and multiple-knockout lines were compared in a hydroponic culture at ammonium concentrations of 0.1 and 3 mM. Under the low-ammonium concentration, in single mutants for each GLN1 gene, there was little effect on growth, whereas the triple mutant for GLN1;1, GLN1;2, and GLN1;3 grew slowly and accumulated ammonium. Under the high-ammonium concentration, the single mutant for GLN1;2 showed 50% decreases in fresh weight and glutamine, whereas the other gln1 single mutants did not show notable changes in the phenotype. The double mutant for GLN1;1 and GLN1;2 showed less growth and a lower glutamine concentration than the single mutant for GLN1;2. Promoter analysis indicated an overlapping expression of GLN1;1 with GLN1;2 in the surface layers of the roots. We thus concluded that: 1) at a low concentration, ammonium was assimilated by GLN1;1, GLN1;2, and GLN1;3, and they were redundant; 2) low-affinity GLN1;2 could contribute to ammonium assimilation at concentrations ranging from 0.1 to 3 mM; and 3) GLN1;1 supported GLN1;2 within the outer cell layers of the root. © The Author 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Brain Cytosolic Phospholipase A2α Mediates Angiotensin II-Induced Hypertension and Reactive Oxygen Species Production in Male Mice.

    Science.gov (United States)

    Song, Chi Young; Khan, Nayaab S; Liao, Francesca-Fang; Wang, Bin; Shin, Ji Soo; Bonventre, Joseph V; Malik, Kafait U

    2018-01-12

    Recently we reported that angiotensin II (Ang II)-induced hypertension is mediated by group IV cytosolic phospholipase A2α (cPLA2α) via production of pro-hypertensive eicosanoids. Since Ang II increases blood pressure via its action in the subfornical organ (SFO), it led us to investigate the expression and possible contribution of cPLA2α to oxidative stress and development of hypertension in this brain area. Adenovirus (Ad)-green fluorescence protein (GFP) cPLA2α short hairpin (sh) RNA (Ad-cPLA2α shRNA) and its control Ad-scrambled shRNA (Ad-Scr shRNA) or Ad-enhanced cyan fluorescence protein cPLA2α DNA (Ad-cPLA2α DNA) and its control Ad-GFP DNA were transduced into SFO of cPLA2α+/+ and cPLA2α-/- male mice, respectively. Ang II (700 ng/kg/min) was infused for 14 days in these mice, and blood pressure was measured by tail-cuff and radio telemetry. cPLA2 activity, reactive oxygen species production, and endoplasmic reticulum stress were measured in the SFO. Transduction of SFO with Ad-cPLA2α shRNA, but not Ad-Scr shRNA in cPLA2α+/+ mice, minimized expression of cPLA2α Ang II-induced cPLA2α activity and oxidative stress in the SFO, blood pressure, and cardiac and renal fibrosis. In contrast, Ad-cPLA2α DNA, but not its control Ad-GFP DNA in cPLA2α-/- mice, restored the expression of cPLA2α, and Ang II-induced increase in cPLA2 activity and oxidative stress in the SFO, blood pressure, cardiac and renal fibrosis. These data suggest that cPLA2α in the SFO is crucial in mediating Ang II-induced hypertension and associated pathogenesis. Therefore, development of selective cPLA2α inhibitors could be useful in treating hypertension and its pathogenesis.

  9. E2F1 interactive with BRCA1 pathway induces HCC two different small molecule metabolism or cell cycle regulation via mitochondrion or CD4+T to cytosol.

    Science.gov (United States)

    Chen, Qingchun; Wang, Lin; Jiang, Minghu; Huang, Juxiang; Jiang, Zhenfu; Feng, Haitao; Ji, Zhili

    2018-02-01

    Breast cancer 1 (BRCA1) and E2F transcription factor 1 (E2F1) are related to metabolism and cell cycle regulation. However, the corresponding mechanism is not clear in HCC. High BRCA1 direct pathway was constructed with 11 molecules from E2F1 feedback-interactive network in HCC by GRNInfer based on 39 Pearson mutual positive corelation CC ≥0.25 molecules with E2F1. Integration of GRNInfer with GO, KEGG, BioCarta, GNF_U133A, UNIGENE_EST, Disease, GenMAPP databases by DAVID and MAS 3.0, E2F1 feedback-interactive BRCA1 indirect mitochondrion to cytosol pathway was identified as upstream LAPTM4B activation, feedback UNG, downstream BCAT1-HIST1H2AD-TK1 reflecting protein, and DNA binding with enrichment of small molecule metabolism; The corresponding BRCA1 indirect membrane to cytosol pathway as upstream CCNB2-NUSAP1 activation, feedback TTK-HIST1H2BJ-CENPF, downstream MCM4-TK1 reflecting ATP, and microtubule binding with enrichment of CD4+T-related cell cycle regulation in HCC. Therefore, we propose that E2F1 interactive with BRCA1 pathway induces HCC two different small molecule metabolism or cell cycle regulation via mitochondrion or CD4+T to cytosol. Knowledge analysis demonstrates our E2F1 feedback-interactive BRCA1 pathway wide disease distribution and reflects a novel common one of tumor and cancer. © 2017 Wiley Periodicals, Inc.

  10. Forced interaction of cell surface proteins with Derlin-1 in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation.

    Science.gov (United States)

    Cho, Sunglim; Lee, Miriam; Jun, Youngsoo

    2013-01-11

    Aberrantly folded proteins in the endoplasmic reticulum (ER) are rapidly removed into the cytosol for degradation by the proteasome via an evolutionarily conserved process termed ER-associated protein degradation (ERAD). ERAD of a subset of proteins requires Derlin-1 for dislocation into the cytosol; however, the molecular function of Derlin-1 remains unclear. Human cytomegalovirus US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules for immune evasion. Because US11 binds to both MHC-I molecules and Derlin-1 via its luminal and transmembrane domains (TMDs), respectively, the major role of US11 has been proposed to simply be delivery of MHC-I molecules to Derlin-1. Here, we directly tested this proposal by generating a hybrid MHC-I molecule, which contains the US11 TMD, and thus can associate with Derlin-1 in the absence of US11. Intriguingly, this MHC-I hybrid was rapidly degraded in a Derlin-1- and proteasome-dependent manner. Similarly, the vesicular stomatitis virus G protein, otherwise expressed at the cell surface, was degraded via Derlin-1-dependent ERAD when its TMD was replaced with that of US11. Thus, forced interaction of cell surface proteins with Derlin-1 is sufficient to induce their degradation via ERAD. Taken together, these results suggest that the main role of US11 is to recruit MHC-I molecules to Derlin-1, which then mediates the dislocation of MHC-I molecules into the cytosol for degradation. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Transfer of a redox-signal through the cytosol by redox-dependent microcompartmentation of glycolytic enzymes at mitochondria and actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Joanna eWojtera-Kwiczor

    2013-01-01

    Full Text Available The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12, GapC plays an important role in glycolysis by providing the cell with ATP and NADH. Interestingly, despite its glycolytic function in the cytosol, GAPDH was reported to possess additional non-glycolytic activities, correlating with its nuclear or cytoskeletal localization in animal cells. In transiently transformed mesophyll protoplasts from Arabidopsis. thaliana colocalization and interaction of the glycolytic enzymes with the mitochondria and with the actin cytoskeleton was visualized by confocal laser scanning microscopy (cLSM using fluorescent protein fusions and by bimolecular fluorescence complementation (BiFC, respectively. Yeast two-hybrid screens, dot-blot overlay assays, and co-sedimentation assays were used to identify potential protein-protein interactions between two cytosolic GAPDH isoforms (GapC1, At3g04120; GapC2, At1g13440 from A. thaliana with the neighbouring glycolytic enzyme, fructose 1,6-bisphosphate aldolase (FBA6, At2g36460, the mitochondrial porin (VDAC3; At5g15090, and actin in vitro. From these experiments, a mitochondrial association is suggested for both glycolytic enzymes, GAPDH and aldolase, which appear to bind to the outer mitochondrial membrane, in a redox-dependent manner. In addition, both glycolytic enzymes were found to bind to F-actin in cosedimentation assays, and lead to bundling of purified rabbit actin, as visualized by cLSM. Actin binding and bundling occurred reversibly under oxidizing conditions. We speculate that such dynamic formation of microcompartments is part of a redox-dependent retrograde signal transduction network for adaptation upon oxidative stress.

  12. [Ca2+]i Elevation and Oxidative Stress Induce KCNQ1 Protein Translocation from the Cytosol to the Cell Surface and Increase Slow Delayed Rectifier (IKs) in Cardiac Myocytes*

    Science.gov (United States)

    Wang, Yuhong; Zankov, Dimitar P.; Jiang, Min; Zhang, Mei; Henderson, Scott C.; Tseng, Gea-Ny

    2013-01-01

    Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca2+]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes. PMID:24142691

  13. [Ca2+]i elevation and oxidative stress induce KCNQ1 protein translocation from the cytosol to the cell surface and increase slow delayed rectifier (IKs) in cardiac myocytes.

    Science.gov (United States)

    Wang, Yuhong; Zankov, Dimitar P; Jiang, Min; Zhang, Mei; Henderson, Scott C; Tseng, Gea-Ny

    2013-12-06

    Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca(2+)]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes.

  14. Structure-activity relationship studies of 1-substituted 3-dodecanoylindole-2-carboxylic acids as inhibitors of cytosolic phospholipase A2-mediated arachidonic acid release in intact platelets.

    Science.gov (United States)

    Griessbach, Klaus; Klimt, Monika; Schulze Elfringhoff, Alwine; Lehr, Matthias

    2002-01-01

    A series of 3-dodecanoylindole-2-carboxylic acid derivatives with varied carboxylic acid substituents at the indole 1-position were synthesized and evaluated for their ability to inhibit arachidonic acid release in human platelets mediated by the cytosolic phospholipase A(2). Structure-activity relationship studies revealed that increasing the polarity of these substituents by the introduction of additional polar groups in the proximity of the carboxylic acid moiety reduced activity. Conformational restriction of the indole-1-carboxylic acid substituents in distinct positions as well as extending the length of these residues led to compounds which did not substantially differ in their potencies.

  15. Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca2+ by mobilization of cellular Ca2+ and activation of Ca2+ influx

    DEFF Research Database (Denmark)

    Bouchelouche, P; Belhage, B; Frandsen, A

    1989-01-01

    The Ca2+ sensitive fluorescent probe, fura-2 has been used to monitor cytosolic free calcium levels in mature primary cultures of cerebellar granule cells during exposure to L-glutamate and other excitatory amino acids: quisqualate (QA) kainate (KA) and N-methyl-D-aspartate (NMDA). Glutamate...... at micromolar concentrations produced a prompt and dose-related increase in the intracellular concentration of free Ca2+, ([Ca2+]i), whereas QA, KA and NMDA had no effect. This increase was also seen in the absence of extracellular Ca2+, suggesting that L-glutamate promotes mobilization of Ca2+ from...

  16. Inhibition of Cytosolic Phospholipase A2α Impairs an Early Step of Coronavirus Replication in Cell Culture.

    Science.gov (United States)

    Müller, Christin; Hardt, Martin; Schwudke, Dominik; Neuman, Benjamin W; Pleschka, Stephan; Ziebuhr, John

    2018-02-15

    Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase A2α (cPLA2α) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLA2α activity, which produces lysophospholipids (LPLs) by cleaving at the sn-2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i) viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLA2α inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA2α activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development.IMPORTANCE Examples of highly conserved RNA virus proteins that qualify as drug targets for broad

  17. Non-invasive in-cell determination of free cytosolic [NAD+]/[NADH] ratios using hyperpolarized glucose show large variations in metabolic phenotypes

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.

    2014-01-01

    Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyse substrate oxidation and as such it plays a key role in various biological processes such as aging, cell ......+]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.......Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyse substrate oxidation and as such it plays a key role in various biological processes such as aging, cell...... death and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labelled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining...

  18. Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

    Science.gov (United States)

    Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

    2015-02-15

    We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. A differential redox regulation of the pathways metabolizing glyceraldehyde-3-phosphate tunes the production of reducing power in the cytosol of plant cells.

    Science.gov (United States)

    Piattoni, Claudia V; Guerrero, Sergio A; Iglesias, Alberto A

    2013-04-12

    Adaptation to aerobic life leads organisms to sense reactive oxygen species and use the signal for coordination of the entire metabolism. Glycolysis in plants is a particular network where specific steps, like oxidation of glyceraldehydes-3-phosphate (Ga3P), are critical in order for it to function. The triose-phosphate can be converted into 3-phosphoglycerate through the phosphorylating Ga3P dehydrogenase (Ga3PDHase, EC 1.2.1.12) producing ATP and NADH, or via the non-phosphorylating enzyme (np-Ga3PDHase; EC 1.2.1.9) generating NADPH. In this work we found redox regulation to be a posttranslational mechanism allowing the fine-tuning of the triose-phosphate fate. Both enzymes were inactivated after oxidation by reactive oxygen and nitrogen species. Kinetic studies determined that Ga3PDHase is marked (63-fold) more sensitive to oxidants than np-Ga3PDHase. Thioredoxin-h reverted the oxidation of both enzymes (although with differences between them), suggesting a physiological redox regulation. The results support a metabolic scenario where the cytosolic triose-phosphate dehydrogenases are regulated under changeable redox conditions. This would allow coordinate production of NADPH or ATP through glycolysis, with oxidative signals triggering reducing power synthesis in the cytosol. The NADPH increment would favor antioxidant responses to cope with the oxidative situation, while the thioredoxin system would positively feedback NADPH production by maintaining np-Ga3PDHase at its reduced active state.

  20. A Differential Redox Regulation of the Pathways Metabolizing Glyceraldehyde-3-Phosphate Tunes the Production of Reducing Power in the Cytosol of Plant Cells

    Directory of Open Access Journals (Sweden)

    Alberto A. Iglesias

    2013-04-01

    Full Text Available Adaptation to aerobic life leads organisms to sense reactive oxygen species and use the signal for coordination of the entire metabolism. Glycolysis in plants is a particular network where specific steps, like oxidation of glyceraldehydes-3-phosphate (Ga3P, are critical in order for it to function. The triose-phosphate can be converted into 3-phosphoglycerate through the phosphorylating Ga3P dehydrogenase (Ga3PDHase, EC 1.2.1.12 producing ATP and NADH, or via the non-phosphorylating enzyme (np-Ga3PDHase; EC 1.2.1.9 generating NADPH. In this work we found redox regulation to be a posttranslational mechanism allowing the fine-tuning of the triose-phosphate fate. Both enzymes were inactivated after oxidation by reactive oxygen and nitrogen species. Kinetic studies determined that Ga3PDHase is marked (63-fold more sensitive to oxidants than np-Ga3PDHase. Thioredoxin-h reverted the oxidation of both enzymes (although with differences between them, suggesting a physiological redox regulation. The results support a metabolic scenario where the cytosolic triose-phosphate dehydrogenases are regulated under changeable redox conditions. This would allow coordinate production of NADPH or ATP through glycolysis, with oxidative signals triggering reducing power synthesis in the cytosol. The NADPH increment would favor antioxidant responses to cope with the oxidative situation, while the thioredoxin system would positively feedback NADPH production by maintaining np-Ga3PDHase at its reduced active state.

  1. Nucleotide exchange between cytosolic ATP and F-actin-bound ADP may be a major energy-utilizing process in unstimulated platelets.

    Science.gov (United States)

    Daniel, J L; Molish, I R; Robkin, L; Holmsen, H

    1986-05-02

    About 40% of the cytosolic ADP of human platelets is tightly bound to protein and the complex is precipitated from the cells by 43% ethanol. We show here that this ADP is bound to F-actin by three criteria (a) copurification with F-actin, (b) specific extraction with water and (c) by specific interaction with DNase I. Platelets contain 0.3 mumol/10(11) cells of this F-actin--ADP complex compared to the total actin content of 0.8 mumol/10(11) cells, which is consistent with the view that actin is maintained in different pools (F-actin--ADP, profilactin, G-actin). In intact platelets the F-actin-bound ADP turns over rapidly and we have determined a turnover rate at 37 degrees C of 0.1 +/- 0.025 s-1 by using a double-labelling procedure. This rapid turnover indicates that F-actin in intact platelets is in a very dynamic state, which may be necessary for rapid responses to stimuli. If it is assumed that the source of the ADP bound to F-actin is cytosolic ATP, the turnover of F-actin ADP measured represents an ATP-consuming process that would account for up to 50% of total ATP consumption in resting platelets.

  2. PDE7B is involved in nandrolone decanoate hydrolysis in liver cytosol and its transcription is up-regulated by androgens in HepG2

    Directory of Open Access Journals (Sweden)

    Emmanuel eStrahm

    2014-05-01

    Full Text Available Most androgenic drugs are available as esters for a prolonged depot action. However the enzymes involved in the hydrolysis of the esters have not been identified. There is one study indicating that PDE7B may be involved in the activation of testosterone enanthate. The aims are to identify the cellular compartments where the hydrolysis of testosterone enanthate and nandrolone decanoate occurs, and to investigate the involvement of PDE7B in the activation. We also determined if testosterone and nandrolone affect the expression of the PDE7B gene. The hydrolysis studies were performed in isolated human liver cytosolic and microsomal preparations with and without specific PDE7B inhibitor. The gene expression was studied in human hepatoma cells (HepG2 exposed to testosterone and nandrolone. We show that PDE7B serves as a catalyst of the hydrolysis of testosterone enanthate and nandrolone decanoate in liver cytosol. The gene expression of PDE7B was significantly induced 3- and 5- fold after 2 hours exposure to 1 µM testosterone enanthate and nandrolone decanoate, respectively. These results show that PDE7B is involved in the activation of esterified nandrolone and testosterone and that the gene expression of PDE7B is induced by supra-physiological concentrations of androgenic drugs.

  3. Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent.

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    Kathleen M Averette

    2009-11-01

    Full Text Available NOD-like receptors (NLRs are a group of cytoplasmic molecules that recognize microbial invasion or 'danger signals'. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT, is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP. The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.

  4. Impaired cellular responses to cytosolic DNA or infection with Listeria monocytogenes and vaccinia virus in the absence of the murine LGP2 protein.

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    Darja Pollpeter

    2011-04-01

    Full Text Available Innate immune signaling is crucial for detection of and the initial response to microbial pathogens. Evidence is provided indicating that LGP2, a DEXH box domain protein related to the RNA recognition receptors RIG-I and MDA5, participates in the cellular response to cytosolic double-stranded DNA (dsDNA. Analysis of embryonic fibroblasts and macrophages from mice harboring targeted disruption in the LGP2 gene reveals that LGP2 can act as a positive regulator of type I IFN and anti-microbial gene expression in response to transfected dsDNA. Results indicate that infection of LGP2-deficient mice with an intracellular bacterial pathogen, Listeria monocytogenes, leads to reduced levels of type I IFN and IL12, and allows increased bacterial growth in infected animals, resulting in greater colonization of both spleen and liver. Responses to infection with vaccinia virus, a dsDNA virus, are also suppressed in cells lacking LGP2, reinforcing the ability of LGP2 to act as a positive regulator of antiviral signaling. In vitro mechanistic studies indicate that purified LGP2 protein does not bind DNA but instead mediates these responses indirectly. Data suggest that LGP2 may be acting downstream of the intracellular RNA polymerase III pathway to activate anti-microbial signaling. Together, these findings demonstrate a regulatory role for LGP2 in the response to cytosolic DNA, an intracellular bacterial pathogen, and a DNA virus, and provide a plausible mechanistic hypothesis as the basis for this activity.

  5. Mechanism of Diphtheria Toxin Catalytic Domain Delivery to the Eukaryotic Cell Cytosol and the Cellular Factors that Directly Participate in the Process

    Science.gov (United States)

    Murphy, John R.

    2011-01-01

    Research on diphtheria and anthrax toxins over the past three decades has culminated in a detailed understanding of their structure function relationships (e.g., catalytic (C), transmembrane (T), and receptor binding (R) domains), as well as the identification of their eukaryotic cell surface receptor, an understanding of the molecular events leading to the receptor-mediated internalization of the toxin into an endosomal compartment, and the pH triggered conformational changes required for pore formation in the vesicle membrane. Recently, a major research effort has been focused on the development of a detailed understanding of the molecular interactions between each of these toxins and eukaryotic cell factors that play an essential role in the efficient translocation of their respective catalytic domains through the trans-endosomal vesicle membrane pore and delivery into the cell cytosol. In this review, I shall focus on recent findings that have led to a more detailed understanding of the mechanism by which the diphtheria toxin catalytic domain is delivered to the eukaryotic cell cytosol. While much work remains, it is becoming increasingly clear that the entry process is facilitated by specific interactions with a number of cellular factors in an ordered sequential fashion. In addition, since diphtheria, anthrax lethal factor and anthrax edema factor all carry multiple coatomer I complex binding motifs and COPI complex has been shown to play an essential role in entry process, it is likely that the initial steps in catalytic domain entry of these divergent toxins follow a common mechanism. PMID:22069710

  6. A krill oil supplemented diet reduces the activities of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes in rats.

    Science.gov (United States)

    Ferramosca, A; Conte, L; Zara, V

    2012-04-01

    The mitochondrial tricarboxylate carrier supplies cytosol with the carbon units necessary for hepatic lipogenesis. The activities of cytosolic acetyl-CoA carboxylase and fatty acid synthetase are therefore strictly connected to the function of mitochondrial tricarboxylate carrier. Dietary polyunsaturated fatty acids (PUFA) are potent modulators of hepatic lipogenesis. In rats fed with a diet enriched with 2.5% krill oil (KO), a novel source of dietary n-3 PUFA, a time-dependent decrease in the activities of the mitochondrial tricarboxylate carrier and of the lipogenic enzymes was found. The KO induced inhibition of hepatic lipogenesis was more pronounced than that found in fish oil (FO)-fed rats, at least at short feeding times. The decrease in the activity of the mitochondrial tricarboxylate carrier caused by KO was due to a reduced expression of the protein. Furthermore, in the KO-fed animals a greater reduction in the levels of hepatic triglycerides and cholesterol was found in comparison to FO-fed rats. © 2011 Blackwell Verlag GmbH.

  7. Fluorescent pH probes, fluorescent proteins, and intrinsic cellular fluorochromes are tools to study cytosolic pH (pHcyt) in mammalian cells.

    Science.gov (United States)

    Martinez, Gloria M.; Gollahon, Lauren S.; Shafer, Keri; Oomman, Sowmini K.; Busch, Christian; Martinez-Zaguilan, Raul

    2001-07-01

    Our understanding of intracellular pH homeostatis in eukaryotic systems has been enhanced since the introduction of carboxyfluorescein diacetate as a useful pH probe more than 20 years ago. BCECF, a derivative of this earlier fluoroprobe has dominated the field. In the past 10 years, SNARF-1 has emerged as an alternative pH probe. Recently, a novel derivative of BCECF, BCPCF has been developed. Green Fluorescent Proteins (GFPs) have also been used recently to monitor pH in a non invasive manner in several cell types. Here, we report that human mammary epithelial cells can be transfected with the gene encoding for cyan (CFP), green (GFP), and yellow (YFP), to study cytosolic pH. The novel red fluorescent protein (DsRed) is not sensitive to pH. Multidrug resistance (MDR) has been associated with altered cytosolic pH homeostasis. We show that experimental maneuvers that decrease pHin enhance the efficacy of chemotherapeutic drugs. We also show that short pulses of UV-B light elicited acidosis in cells, as evaluated by ratio ion cell imaging, and confocal/spectral imaging microscopy. During the course of these experiments we noticed that cells exhibit intrinsic fluorochromes that can be used to monitor pH in living cells.

  8. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    Science.gov (United States)

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Retargeting Clostridium difficile Toxin B to Neuronal Cells as a Potential Vehicle for Cytosolic Delivery of Therapeutic Biomolecules to Treat Botulism

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    Greice Krautz-Peterson

    2012-01-01

    Full Text Available Botulinum neurotoxins (BoNTs deliver a protease to neurons which can cause a flaccid paralysis called botulism. Development of botulism antidotes will require neuronal delivery of agents that inhibit or destroy the BoNT protease. Here, we investigated the potential of engineering Clostridium difficile toxin B (TcdB as a neuronal delivery vehicle by testing two recombinant TcdB chimeras. For AGT-TcdB chimera, an alkyltransferase (AGT was appended to the N-terminal glucosyltransferase (GT of TcdB. Recombinant AGT-TcdB had alkyltransferase activity, and the chimera was nearly as toxic to Vero cells as wild-type TcdB, suggesting efficient cytosolic delivery of the AGT/GT fusion. For AGT-TcdB-BoNT/A-Hc, the receptor-binding domain (RBD of TcdB was replaced by the equivalent RBD from BoNT/A (BoNT/A-Hc. AGT-TcdB-BoNT/A-Hc was >25-fold more toxic to neuronal cells and >25-fold less toxic to Vero cells than AGT-TcdB. Thus, TcdB can be engineered for cytosolic delivery of biomolecules and improved targeting of neuronal cells.

  10. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    Science.gov (United States)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  11. Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

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    Shadab Anwar

    Full Text Available Iron-Sulfur (Fe-S proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1 protein and Nucleotide binding protein 35 (Nbp35. In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151 of Nbp35 and (G5-V6, M34-D39 and G46-A52 of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins

  12. Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

    Science.gov (United States)

    Anwar, Shadab; Dikhit, Manas Ranjan; Singh, Krishn Pratap; Kar, Rajiv Kumar; Zaidi, Amir; Sahoo, Ganesh Chandra; Roy, Awadh Kishore; Nozaki, Tomoyoshi; Das, Pradeep; Ali, Vahab

    2014-01-01

    Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any

  13. SCFSLF-mediated cytosolic degradation of S-RNase is required for cross-pollen compatibility in S-RNase-based self-incompatibility in Petunia hybrida

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    Yongbiao eXue

    2014-07-01

    Full Text Available Many flowering plants adopt self-incompatibility (SI to maintain their genetic diversity. In species of Solanaceae, Plantaginaceae and Rosaceae, SI is genetically controlled by a single S-locus with multiple haplotypes. The S-locus has been shown to encode S-RNases expressed in pistil and multiple SLF (S-locus F-box proteins in pollen controlling the female and male specificity of SI, respectively. S-RNases appear to function as a cytotoxin to reject self-pollen. In addition, SLFs have been shown to form SCF (SKP1/Cullin1/F-box complexes to serve as putative E3 ubiquitin ligase to interact with S-RNases. Previously, two different mechanisms, the S-RNase degradation and the S-RNase compartmentalization, have been proposed as the restriction mechanisms of S-RNase cytotoxicity allowing compatible pollination. In this study, we have provided several lines of evidence in support of the S-RNase degradation mechanism by a combination of cellular, biochemical and molecular biology approaches. First, both immunogold labeling and subcellular fractionation assays showed that two key pollen SI factors, PhSLF-S3L and PhSSK1 (SLF-interacting SKP1-like1 from Petunia hybrida, a Solanaceous species, are co-localized in cytosols of both pollen grains and tubes. Second, PhS3L-RNases are mainly detected in the cytosols of both self and non-self pollen tubes after pollination. Third, we found that both PhS3-RNases and PhS3L-RNases directly interact with PhSLF-S3L by yeast two-hybrid and co-immunoprecipitation assays. Fourth, S-RNases are specifically degraded in compatible pollen tubes by non-self SLF action. Taken together, our results demonstrate that SCFSLF-mediated non-self S-RNase degradation occurs in the cytosol of pollen tube through the ubiquitin/26S proteasome system serving as the major mechanism to neutralize S-RNase cytotoxicity during compatible pollination in P. hybrida.

  14. Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

    Energy Technology Data Exchange (ETDEWEB)

    García-Sevillano, M.A.; García-Barrera, T. [Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007 (Spain); Research Center on Health and Environment (CYSMA), University of Huelva (Spain); International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain); Navarro, F. [International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain); Department of Environmental Biology and Public Health, Cell Biology, Faculty of Experimental Sciences, University of Huelva, Campus El Carmen, Huelva 21007 (Spain); Gómez-Ariza, J.L., E-mail: ariza@uhu.es [Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007 (Spain); Research Center on Health and Environment (CYSMA), University of Huelva (Spain); International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain)

    2014-09-09

    Highlights: • Identification and quantification of Cu,Zn-superoxide dismutase in mice hepatic cells. • IDA-ICP-MSis applied to obtain a high degree of accuracy, precision and sensibility. • This methodology reduces the time of analysis and avoids clean-up procedures. • The application of this method to Hg-exposed mice reveals perturbations in Cu,Zn-SOD. - Abstract: In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min

  15. The compartmentalisation of phosphorylated free oligosaccharides in cells from a CDG Ig patient reveals a novel ER-to-cytosol translocation process.

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    Delphine Peric

    Full Text Available BACKGROUND: Biosynthesis of the dolichol linked oligosaccharide (DLO required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man(5GlcNAc(2-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc(3Man(9GlcNAc(2-PP-dolichol. After transfer of Glc(3Man(9GlcNAc(2 onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P. Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation. METHODS AND PRINCIPAL FINDINGS: In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man(7GlcNAc(2-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man(7GlcNAc(2-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man(7GlcNAc(2-P appears in the cytosol without detectable generation of ER luminal Man(7GlcNAc(2-P. CONCLUSIONS AND SIGNIFICANCE: The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that

  16. A mutational analysis of the cytosolic domain of the tomato Cf-9 disease-resistance protein shows that membrane-proximal residues are important for Avr9-dependent necrosis.

    Science.gov (United States)

    Chakrabarti, Apratim; Velusamy, Thilaga; Tee, Choon Yang; Jones, David A

    2016-05-01

    The tomato Cf-9 gene encodes a membrane-anchored glycoprotein that imparts race-specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N-terminal half of the extracellular leucine-rich repeat (eLRR) domain of the Cf-9 protein determines its specificity for Avr9, the C-terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf-9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9-dependent necrosis triggered by Cf-9. Our results indicate that the membrane-proximal region of the cytosolic domain of Cf-9 plays an important role in Cf-9-mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf-9 function. An alanine mutation of T835 had no effect on Cf-9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf-9 function. We therefore postulate that phosphorylation/de-phosphorylation of T835 could act as a molecular switch to determine whether Cf-9 is in a primed or inactive state. Yeast two-hybrid analysis was used to show that the cytosolic domain of Cf-9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline-induced kink in the membrane-proximal region of the cytosolic domain of Cf-9 may be important for interaction with VAP27, which may, in turn, be important for Cf-9 function. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  17. Ancient Plant Glyoxylate/Succinic Semialdehyde Reductases: GLYR1s Are Cytosolic, Whereas GLYR2s Are Localized to Both Mitochondria and Plastids

    Directory of Open Access Journals (Sweden)

    Barry J. Shelp

    2017-04-01

    Full Text Available Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (GLYR1 and GLYR2 are considered to be involved in detoxifying harmful aldehydes, thereby preserving plant health during exposure to various abiotic stresses. Phylogenetic analysis revealed that the two GLYR isoforms appeared in the plant lineage prior to the divergence of the Chlorophyta and Streptophyta, which occurred approximately 750 million years ago. Green fluorescent protein fusions of apple (Malus x domestica Borkh., rice (Oryza sativa L. and Arabidopsis thaliana [L.] Heynh GLYRs were transiently expressed in tobacco (Nicotiana tabaccum L. suspension cells or Arabidopsis protoplasts, as well in methoxyfenozide-induced, stably transformed Arabidopsis seedlings. The localization of apple GLYR1 confirmed that this isoform is cytosolic, whereas apple, rice and Arabidopsis GLYR2s were localized to both mitochondria and plastids. These findings highlight the potential involvement of GLYRs within distinct compartments of the plant cell.

  18. Isolation and characterization of binding proteins for retinol from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens.

    Science.gov (United States)

    Rao, M R; Prasad, V R; Padmanaban, G; Ganguly, J

    1979-01-01

    Protein fractions that bind retinol were isolated from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens. The proteins isolated from the three sources showed similar elution profiles on chromatography through Sephadex G-75 and G-50 columns, and comparable mobility during electrophoresis on sodium dodecyl sulphate/polyacrylamide gels. Their molecular weights were calculated to be around 14500. When oviducts from vitamin A-depleted and vitamin A-repleted immature chicks given oestrogen injections for 6 consecutive days were incubated with [3H]retinyl acetate, uptake of the radioactivity in the nuclei of the vitamin A-depleted tissue was severalfold higher than that in the nuclei from the vitamin A-repleted tissue. Images Fig. 3. PMID:575485

  19. Mitochondrial, acidic, and cytosolic pHs determination by ³¹P NMR spectroscopy: design of new sensitive targeted pH probes.

    Science.gov (United States)

    Culcasi, Marcel; Thétiot-Laurent, Sophie; Atteia, Ariane; Pietri, Sylvia

    2015-01-01

    (31)P nuclear magnetic resonance (NMR) is a unique technique to monitor noninvasively the energetics of living systems at real time through the detection of a variety of phosphorylated metabolites. Using adequately designed α-aminophosphonates as external probes, we have shown earlier that (31)P NMR can also give access simultaneously to the accurate pH of cytosolic and acidic compartments in normal and stressed cultured cells or isolated perfused organs, a feature that was not possible using endogenous inorganic phosphate as the probe. More recently, we obtained a series of derivatives of these new pH probes that incorporate a triphenylphosphonium cation as a specific vector to the mitochondrion. Here, we describe the synthesis, (31)P NMR pH titrating properties in buffers, and application in cultures of the green alga Chlamydomonas reinhardtii of two of these mitochondria-targeted pH probes in comparison with one nonvectorized, yet still informative α-aminophosphonate.

  20. Cytosolic Glutamine Synthetase is Important for Photosynthetic Efficiency and Water Use Efficiency in Potato as Revealed by High Throughput Sequencing QTL analysis

    DEFF Research Database (Denmark)

    Kaminski, Kacper Piotr; Sørensen, Kirsten Kørup; Andersen, Mathias Neumann

    2015-01-01

    efficiency (WUE) of potato is important, but has been limited by technical difficulties in assessing the trait on individual plants and thus is poorly understood. In this study, a mapping population of potatoes has been robustly phenotyped, and considerable variation in WUE under well-watered conditions......Potato (Solanum tuberosum L.) closes its stomata at relatively low soil water deficits frequently encountered in normal field conditions resulting in unnecessary annual yield losses and extensive use of artificial irrigation. Therefore, unraveling the genetics underpinning variation in water use...... isoforms of cytosolic glutamine synthase were located in the QTL at chromosome 1 suggesting a major contribution of this enzyme to photosynthetic efficiency and thus WUE in potato. Indeed, Glutamine synthetase enzyme activity of leaf extracts was measured and found to be correlated with contrasting WUE...

  1. NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi.

    Science.gov (United States)

    Nývltová, Eva; Šuták, Robert; Harant, Karel; Šedinová, Miroslava; Hrdy, Ivan; Paces, Jan; Vlček, Čestmír; Tachezy, Jan

    2013-04-30

    In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the α-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an ε-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments.

  2. Forkhead box protein O3 transcription factor negatively regulates autophagy in human cancer cells by inhibiting forkhead box protein O1 expression and cytosolic accumulation.

    Directory of Open Access Journals (Sweden)

    Wan Long Zhu

    Full Text Available FoxO proteins are important regulators in cellular metabolism and are recognized to be nodes in multiple signaling pathways, most notably those involving PI3K/AKT and mTOR. FoxO proteins primarily function as transcription factors, but recent study suggests that cytosolic FoxO1 participates in the regulation of autophagy. In the current study, we find that cytosolic FoxO1 indeed stimulates cellular autophagy in multiple cancer cell lines, and that it regulates not only basal autophagy but also that induced by rapamycin and that in response to nutrient deprivation. These findings illustrate the importance of FoxO1 in cell metabolism regulation independent of its transcription factor function. In contrast to FoxO1, we find the closely related FoxO3a is a negative regulator of autophagy in multiple cancer cell lines, a previously unrecognized function for this protein, different from its function in benign fibroblast and muscle cells. The induction of autophagy by the knockdown of FoxO3a was found not to be mediated through the suppression of mTORC1 signaling; rather, the regulatory role of FoxO3a on autophagy was determined to be through its ability to transcriptionally suppress FoxO1. This complicated interplay of FoxO1 and FoxO3a suggests a complex checks- and balances-relationship between FoxO3a and FoxO1 in regulating autophagy and cell metabolism.

  3. Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a cGAS/STING-mediated cytosolic DNA-sensing pathway.

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    Peihong Dai

    2014-04-01

    Full Text Available Modified vaccinia virus Ankara (MVA is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs, which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs, but not in plasmacytoid dendritic cells (pDCs. Transcription factors IRF3 (IFN regulatory factor 3 and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1, are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase. MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1 and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.

  4. A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel.

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    Thompson, Jill L; Shuttleworth, Trevor J

    2012-01-01

    The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.

  5. The Campylobacter jejuni CiaC virulence protein is secreted from the flagellum and delivered to the cytosol of host cells

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    Jason eNeal-McKinney

    2012-03-01

    Full Text Available Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute C. jejuni-mediated disease (campylobacteriosis involves C. jejuni invasion of host epithelial cells using a set of virulence proteins known as the Campylobacter invasion antigens (Cia. The genes encoding the Cia proteins are up-regulated upon co-culture of C. jejuni with epithelial cells. One of the Cia proteins, CiaC, is required for maximal invasion of host cells by C. jejuni. Previous work has also revealed that CiaC is, in part, responsible for host cell cytoskeletal rearrangements that result in membrane ruffling. This study was performed to test the hypothesis that CiaC is delivered to the cytosol of host cells. To detect the delivery of CiaC into cultured epithelial cells, we used the adenylate cyclase domain (ACD of Bordetella pertussis CyaA as a reporter. In this study, we found that export and delivery of the C. jejuni Cia proteins into human INT 407 epithelial cells required a functional flagellar hook complex composed of FlgE, FlgK, and FlgL. Assays performed with bacterial culture supernatants supported the hypothesis that CiaC delivery requires bacteria-host cell contact. We also found that that CiaC was delivered to host cells by cell-associated (bound bacteria, as judged by experiments performed with inhibitors that specifically target the cell signaling pathways utilized by C. jejuni for cell invasion. Interestingly, the C. jejuni flgL mutant, which is incapable of exporting and delivering the Cia proteins, did not induce INT 407 cell membrane ruffles. Complementation of the flgL mutant with plasmid-encoded flgL restored the motility and membrane ruffling. These data support the hypothesis that the C. jejuni Cia proteins, which are exported from the flagellum, are delivered to the cytosol of host cells.

  6. Excessive signal transduction of gain-of-function variants of the calcium-sensing receptor (CaSR are associated with increased ER to cytosol calcium gradient.

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    Marianna Ranieri

    Full Text Available In humans, gain-of-function mutations of the calcium-sensing receptor (CASR gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA and reducing expression of Plasma Membrane Calcium-ATPase (PMCA. Wild-type CaSR (hCaSR-wt and its gain-of-function (hCaSR-R990G; hCaSR-N124K variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate receptor inputs to cell function.

  7. Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz).

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    Xu, Jia; Yang, Jun; Duan, Xiaoguang; Jiang, Yueming; Zhang, Peng

    2014-08-05

    Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H₂O₂, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H₂O₂ synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress.

  8. Primary Role for Toll-Like Receptor-Driven Tumor Necrosis Factor Rather than Cytosolic Immune Detection in Restricting Coxiella burnetii Phase II Replication within Mouse Macrophages

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    Bradley, William P.; Boyer, Mark A.; Nguyen, Hieu T.; Birdwell, L. Dillon; Yu, Janet; Ribeiro, Juliana M.; Roy, Craig R.

    2016-01-01

    Coxiella burnetii replicates within permissive host cells by employing a Dot/Icm type IV secretion system (T4SS) to translocate effector proteins that direct the formation of a parasitophorous vacuole. C57BL/6 mouse macrophages restrict the intracellular replication of the C. burnetii Nine Mile phase II (NMII) strain. However, eliminating Toll-like receptor 2 (TLR2) permits bacterial replication, indicating that the restriction of bacterial replication is immune mediated. Here, we examined whether additional innate immune pathways are employed by C57BL/6 macrophages to sense and restrict NMII replication. In addition to the known role of TLR2 in detecting and restricting NMII infection, we found that TLR4 also contributes to cytokine responses but is not required to restrict bacterial replication. Furthermore, the TLR signaling adaptors MyD88 and Trif are required for cytokine responses and restricting bacterial replication. The C. burnetii NMII T4SS translocates bacterial products into C57BL/6 macrophages. However, there was little evidence of cytosolic immune sensing of NMII, as there was a lack of inflammasome activation, T4SS-dependent cytokine responses, and robust type I interferon (IFN) production, and these pathways were not required to restrict bacterial replication. Instead, endogenous tumor necrosis factor (TNF) produced upon TLR sensing of C. burnetii NMII was required to control bacterial replication. Therefore, our findings indicate a primary role for TNF produced upon immune detection of C. burnetii NMII by TLRs, rather than cytosolic PRRs, in enabling C57BL/6 macrophages to restrict bacterial replication. PMID:26787725

  9. Astrocytic energy metabolism and glutamate formation--relevance for 13C-NMR spectroscopy and importance of cytosolic/mitochondrial trafficking.

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    Hertz, Leif

    2011-12-01

    Glutamate plays a double role in (13)C-nuclear magnetic resonance (NMR) spectroscopic determination of glucose metabolism in the brain. Bidirectional exchange between initially unlabeled glutamate and labeled α-ketoglutarate, formed from pyruvate via pyruvate dehydrogenase (PDH), indicates the rate of energy metabolism in the tricarboxylic acid (V(TCA)) cycle in neurons (V(PDH, n)) and, with additional computation, also in astrocytes (V(PDH, g)), as confirmed using the astrocyte-specific substrate [(13)C]acetate. Formation of new molecules of glutamate during increased glutamatergic activity occurs only in astrocytes by combined pyruvate carboxylase (V(PC)) and astrocytic PDH activity. V(PDH, g) accounts for ~15% of total pyruvate metabolism in the brain cortex, and V(PC) accounts for another ~10%. Since both PDH-generated and PC-generated pyruvates are needed for glutamate synthesis, ~20/25 (80%) of astrocytic pyruvate metabolism proceed via glutamate formation. Net transmitter glutamate [γ-aminobutyric acid (GABA)] formation requires transfer of newly synthesized α-ketoglutarate to the astrocytic cytosol, α-ketoglutarate transamination to glutamate, amidation to glutamine, glutamine transfer to neurons, its hydrolysis to glutamate and glutamate release (or GABA formation). Glutamate-glutamine cycling, measured as glutamine synthesis rate (V(cycle)), also transfers previously released glutamate/GABA to neurons after an initial astrocytic accumulation and measures predominantly glutamate signaling. An empirically established ~1/1 ratio between glucose metabolism and V(cycle) may reflect glucose utilization associated with oxidation/reduction processes during glutamate production, which together with associated transamination processes are balanced by subsequent glutamate oxidation after cessation of increased signaling activity. Astrocytic glutamate formation and subsequent oxidative metabolism provide large amounts of adenosine triphosphate used for

  10. Fumarate and cytosolic pH as modulators of the synthesis or consumption of C(4) organic acids through NADP-malic enzyme in Arabidopsis thaliana.

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    Arias, Cintia Lucía; Andreo, Carlos Santiago; Drincovich, María Fabiana; Gerrard Wheeler, Mariel Claudia

    2013-02-01

    Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. Among the enzymes that metabolize these compounds, one of the most important ones is malic enzyme (ME). A. thaliana contains four malic enzymes (NADP-ME 1-4) to catalyze the reversible oxidative decarboxylation of malate in the presence of NADP. NADP-ME2 is the only one located in the cell cytosol of all Arabidopsis organs providing most of the total NADP-ME activity. In the present work, the regulation of this key enzyme by fumarate was investigated by kinetic assays, structural analysis and a site-directed mutagenesis approach. The final effect of this metabolite on NADP-ME2 forward activity not only depends on fumarate and substrate concentrations but also on the pH of the reaction medium. Fumarate produced an increase in NADP-ME2 activity by binding to an allosteric site. However at higher concentrations, fumarate caused a competitive inhibition, excluding the substrate malate from binding to the active site. The characterization of ME2-R115A mutant, which is not activated by fumarate, confirms this hypothesis. In addition, the reverse reaction (reductive carboxylation of pyruvate) is also modulated by fumarate, but in a different way. The results indicate pH-dependence of the fumarate modulation with opposite behavior on the two activities analyzed. Thereby, the coordinated action of fumarate over the direct and reverse reactions would allow a precise and specific modulation of the metabolic flux through this enzyme, leading to the synthesis or degradation of C(4) compounds under certain conditions. Thus, the physiological context might be exerting an accurate control of ME activity in planta, through changes in metabolite and substrate concentrations and cytosolic pH.

  11. The stress response against denatured proteins in the deletion of cytosolic chaperones SSA1/2 is different from heat-shock response in Saccharomyces cerevisiae

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    Matsumoto, Rena; Akama, Kuniko; Rakwal, Randeep; Iwahashi, Hitoshi

    2005-01-01

    Background A yeast strain lacking the two genes SSA1 and SSA2, which encode cytosolic molecular chaperones, acquires thermotolerance as well as the mild heat-shocked wild-type yeast strain. We investigated the genomic response at the level of mRNA expression to the deletion of SSA1/2 in comparison with the mild heat-shocked wild-type using cDNA microarray. Results Yeast cDNA microarray analysis revealed that genes involved in the stress response, including molecular chaperones, were up-regulated in a similar manner in both the ssa1/2 deletion mutant and the mild heat-shocked wild-type. Genes involved in protein synthesis were up-regulated in the ssa1/2 deletion mutant, but were markedly suppressed in the mild heat-shocked wild-type. The genes involved in ubiquitin-proteasome protein degradation were also up-regulated in the ssa1/2 deletion mutant, whereas the unfolded protein response (UPR) genes were highly expressed in the mild heat-shocked wild-type. RT-PCR confirmed that the genes regulating protein synthesis and cytosolic protein degradation were up-regulated in the ssa1/2 deletion mutant. At the translational level, more ubiquitinated proteins and proteasomes were detected in the ssa1/2 deletion mutant, than in the wild-type, confirming that ubiquitin-proteasome protein degradation was up-regulated by the deletion of SSA1/2. Conclusion These results suggest that the mechanism for rescue of denatured proteins in the ssa1/2 deletion mutant is different from that in the mild heat-shocked wild-type: Activated protein synthesis in the ssa1/2 deletion mutant supplies a deficiency of proteins by their degradation, whereas mild heat-shock induces UPR. PMID:16209719

  12. Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation in Chlamydomonas.

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    Wu, Qiong; Gao, Kang; Zheng, Shuzhi; Zhu, Xin; Liang, Yinwen; Pan, Junmin

    2018-01-29

    Calcium has been implicated in the motility, assembly, disassembly, and deflagellation of the eukaryotic flagellum or cilium (exchangeable terms). Calmodulin (CaM) is known to be critical for flagellar motility; however, it is unknown whether and how CaM is involved in other flagella-related activities. We have studied CaM in Chlamydomonas, a widely used organism for ciliary studies. CaM is present in the cell body and the flagellum, with enrichment in the basal body region. Loss of CaM causes shortening of the nucleus basal body connector and impairs flagellar motility and assembly but not flagellar disassembly. Moreover, the cam mutant is defective in pH shock-induced deflagellation. The mutant deflagellates, however, upon mechanical shearing and treatment with mastoparan or detergent undergo permeabilization in the presence of calcium, indicating the cam mutant is defective in elevations of cytosolic calcium induced by pH shock, rather than by the deflagellation machinery. Indeed, the cam mutant fails to produce inositol 1,4,5-trisphosphate. Biochemical and genetic analysis showed that CaM does not directly activate PLC. Furthermore, CaM interacts with ADF1, a transient receptor channel that functions in acid-induced calcium entry. Thus, CaM is a critical regulator of flagellar activities especially those involved in modulating calcium homeostasis during acidic stress.-Wu, Q., Gao, K., Zheng, S., Zhu, X., Liang, Y., Pan, J. Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation in Chlamydomonas.

  13. Modeling and simulation of aggregation of membrane protein LAT with molecular variability in the number of binding sites for cytosolic Grb2-SOS1-Grb2.

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    Ambarish Nag

    Full Text Available The linker for activation of T cells (LAT, the linker for activation of B cells (LAB, and the linker for activation of X cells (LAX form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1:1 and 2:1 complexes with the guanine nucleotide exchange factor SOS1. The 2:1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate. We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.

  14. ATRA-induced HL-60 myeloid leukemia cell differentiation depends on the CD38 cytosolic tail needed for membrane localization, but CD38 enzymatic activity is unnecessary.

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    Congleton, Johanna; Jiang, Hong; Malavasi, Fabio; Lin, Hening; Yen, Andrew

    2011-04-15

    Leukocyte antigen CD38 expression is an early marker of all-trans retinoic acid (ATRA) stimulated differentiation in the leukemic cell line HL-60. It promotes induced myeloid maturation when overexpressed, whereas knocking it down is inhibitory. It is a type II membrane protein with an extracellular C-terminal enzymatic domain with NADase/NADPase and ADPR cyclase activity and a short cytoplasmic N-terminal tail. Here we determined whether CD38 enzymatic activity or the cytoplasmic tail is required for ATRA-induced differentiation. Neither a specific CD38 ectoenzyme inhibitor nor a point mutation that cripples enzymatic activity (CD38 E226Q) diminishes ATRA-induced differentiation or G1/0 arrest. In contrast a cytosolic deletion mutation (CD38 Δ11-20) prevents membrane expression and inhibits differentiation and G1/0 arrest. These results may be consistent with disrupting the function of critical molecules necessary for membrane-expressed CD38 signal transduction. One candidate molecule is the Src family kinase Fgr, which failed to undergo ATRA-induced upregulation in CD38 Δ11-20 expressing cells. Another is Vav1, which also showed only basal expression after ATRA treatment in CD38 Δ11-20 expressing cells. Therefore, the ability of CD38 to propel ATRA-induced myeloid differentiation and G1/0 arrest is unimpaired by loss of its ectoenzyme activity. However a cytosolic tail deletion mutation disrupted membrane localization and inhibited differentiation. ATRA-induced differentiation thus does not require the CD38 ectoenzyme function, but is dependent on a membrane receptor function. Copyright © 2010. Published by Elsevier Inc.

  15. Excessive Signal Transduction of Gain-of-Function Variants of the Calcium-Sensing Receptor (CaSR) Are Associated with Increased ER to Cytosol Calcium Gradient

    Science.gov (United States)

    Di Mise, Annarita; Vezzoli, Giuseppe; Soldati, Laura; Svelto, Maria; Valenti, Giovanna

    2013-01-01

    In humans, gain-of-function mutations of the calcium-sensing receptor (CASR) gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA) and reducing expression of Plasma Membrane Calcium-ATPase (PMCA). Wild-type CaSR (hCaSR-wt) and its gain-of-function (hCaSR-R990G; hCaSR-N124K) variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate) receptor inputs to cell function. PMID:24244430

  16. Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation.

    Science.gov (United States)

    Köllmer, Ireen; Novák, Ondřej; Strnad, Miroslav; Schmülling, Thomas; Werner, Tomáš

    2014-05-01

    Degradation of the plant hormone cytokinin is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. The Arabidopsis thaliana genome encodes seven CKX proteins which differ in subcellular localization and substrate specificity. Here we analyze the CKX7 gene, which to the best of our knowledge has not yet been studied. pCKX7:GUS expression was detected in the vasculature, the transmitting tissue and the mature embryo sac. A CKX7-GFP fusion protein localized to the cytosol, which is unique among all CKX family members. 35S:CKX7-expressing plants developed short, early terminating primary roots with smaller apical meristems, contrasting with plants overexpressing other CKX genes. The vascular bundles of 35S:CKX7 primary roots contained only protoxylem elements, thus resembling the wol mutant of the CRE1/AHK4 receptor gene. We show that CRE1/AHK4 activity is required to establish the CKX7 overexpression phenotype. Several cytokinin metabolites, in particular cis-zeatin (cZ) and N-glucoside cytokinins, were depleted stronger in 35S:CKX7 plants compared with plants overexpressing other CKX genes. Interestingly, enhanced protoxylem formation together with reduced primary root growth was also found in the cZ-deficient tRNA isopentenyltransferase mutant ipt2,9. However, different cytokinins were similarly efficient in suppressing 35S:CKX7 and ipt2,9 vascular phenotypes. Therefore, we hypothesize that the pool of cytosolic cytokinins is particularly relevant in the root procambium where it mediates the differentiation of vascular tissues through CRE1/AHK4. Taken together, the distinct consequences of CKX7 overexpression indicate that the cellular compartmentalization of cytokinin degradation and substrate preference of CKX isoforms are relevant parameters that define the activities of the hormone. © 2014 The Authors. The Plant Journal © 2014 John Wiley & Sons Ltd.

  17. Overexpression of a cytosolic abiotic stress responsive universal stress protein (SbUSP mitigates salt and osmotic stress in transgenic tobacco plants

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    Pushpika eUdawat

    2016-04-01

    Full Text Available The Universal Stress Protein (USP is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologues of intron less SbUSP gene which encodes for salt and osmotic responsive universal stress protein. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control (wild type and vector control plants under different abiotic stress condition. Transgenic lines (T1 exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability and lower electrolyte leakage and lipid peroxidation (malondialdehyde content under stress treatments than control (WT and VC plants. Lower accumulation of H2O2 and O2- radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis (PCA exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant.

  18. DNA adducts from nitroreduction of 2,7-dinitrofluorene, a mammary gland carcinogen, catalyzed by rat liver or mammary gland cytosol.

    Science.gov (United States)

    Ritter, Clare L; Culp, Sandra J; Freeman, James P; Marques, M Matilde; Beland, Frederick A; Malejka-Giganti, Danuta

    2002-04-01

    Nitrofluorenes are mutagenic and carcinogenic environmental pollutants arising chiefly from combustion of fossil fuels. Nitro aromatic compounds undergo nitroreduction to N-hydroxy arylamines that bind to DNA directly or after O-esterification. This study analyzes the DNA binding and adducts from the in vitro nitroreduction of 2,7-dinitrofluorene (2,7-diNF), a potent mammary carcinogen in the rat. Potential adduct(s) of 2,7-diNF was (were) generated by reduction of 2-nitroso-7-NF with ascorbate/H(+) in the presence of calf thymus DNA. The major adduct was characterized by HPLC/ESI/MS and (1)H NMR spectrometry as N-(deoxyguanosin-8-yl)-2-amino-7-NF, and a minor one was determined by HPLC/ESI/MS to be a deoxyadenosine adduct of 2-amino-7-NF. Products from enzymatic nitroreduction were monitored by HPLC and DNA adduct formation by (32)P-postlabeling. Xanthine oxidase/hypoxanthine-catalyzed nitroreduction of 2,7-diNF, 2-nitrofluorene (2-NF), and 1-nitropyrene (1-NP) yielded the respective amines to similar extents (30-50%). However, the level of the major adducts ( approximately 0.15/10(6) nucleotides) from 2-NF [N-(deoxyguanosin-8-yl)-2-aminofluorene] and 2,7-diNF [N-(deoxyguanosin-8-yl)-2-amino-7-NF] was 30 adducts/10(6) nucleotides, levels comparable to those from 1,6-dinitropyrene and 4- or 49-fold greater than the respective levels without acetyl CoA. Recovery of 2-nitroso-7-NF and 2-amino-7-NF from cytosol-catalyzed reduction of 2,7-diNF indicated nitroreduction and an N-hydroxy arylamine intermediate. Likewise, the presence of 2-acetylamino-7-NF indicated that reactivity with acyltransferase(s) was not prevented by the nitro group at C7. These data are consistent with activation of 2,7-diNF via nitroreduction to the N-hydroxy arylamine and acetyl CoA-dependent O-acetylation of the latter to bind to DNA. Enzymatic nitroreduction of 2,7-diNF was greatly enhanced by 9-oxidation. The nitroreduction of either 9-oxo-2,7-diNF or 9-hydroxy-2,7-diNF catalyzed by liver

  19. A mutation in the cytosolic O-acetylserine (thiol lyase induces a genome-dependent early leaf death phenotype in Arabidopsis

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    Schippers Jos HM

    2010-04-01

    Full Text Available Abstract Background Cysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol lyase (OAS-TL catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified. Results The seedling lethal phenotype of the Arabidopsis onset of leaf death3-1 (old3-1 mutant is due to a point mutation in the OAS-A1 gene, encoding the cytosolic OAS-TL. The mutation causes a single amino acid substitution from Gly162 to Glu162, abolishing old3-1 OAS-TL activity in vitro. The old3-1 mutation segregates as a monogenic semi-dominant trait when backcrossed to its wild type accession Landsberg erecta (Ler-0 and the Di-2 accession. Consistent with its semi-dominant behaviour, wild type Ler-0 plants transformed with the mutated old3-1 gene, displayed the early leaf death phenotype. However, the old3-1 mutation segregates in an 11:4:1 (wild type: semi-dominant: mutant ratio when backcrossed to the Colombia-0 and Wassilewskija accessions. Thus, the early leaf death phenotype depends on two semi-dominant loci. The second locus that determines the old3-1 early leaf death phenotype is referred to as odd-ler (for old3 determinant in the Ler accession and is located on chromosome 3. The early leaf death phenotype is temperature dependent and is associated with increased expression of defence-response and oxidative-stress marker genes. Independent of the presence of the odd-ler gene, OAS-A1 is involved in maintaining sulphur and thiol levels and is required for resistance against cadmium stress. Conclusions The cytosolic OAS-TL is involved in maintaining organic sulphur levels. The old3-1 mutation causes genome-dependent and independent phenotypes and uncovers a novel function for the mutated OAS-TL in cell

  20. Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells.

    Science.gov (United States)

    Sun, Wen; Wu, Xiaxia; Gao, Hongwei; Yu, Jie; Zhao, Wenwen; Lu, Jin-Jian; Wang, Jinhua; Du, Guanhua; Chen, Xiuping

    2017-07-01

    Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca2+) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium

  1. Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models.

    Science.gov (United States)

    Wen, Li; Voronina, Svetlana; Javed, Muhammad A; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V; Criddle, David N; Sutton, Robert

    2015-08-01

    Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis

  2. HbNIN2, a cytosolic alkaline/neutral-invertase, is responsible for sucrose catabolism in rubber-producing laticifers of Hevea brasiliensis (para rubber tree).

    Science.gov (United States)

    Liu, Shujin; Lan, Jixian; Zhou, Binhui; Qin, Yunxia; Zhou, Yihua; Xiao, Xiaohu; Yang, Jianghua; Gou, Jiqing; Qi, Jiyan; Huang, Yacheng; Tang, Chaorong

    2015-04-01

    In Hevea brasiliensis, an alkaline/neutral invertase (A/N-Inv) is responsible for sucrose catabolism in latex (essentially the cytoplasm of rubber-producing laticifers, the source of natural rubber) and implicated in rubber yield. However, neither the gene encoding this enzyme nor its molecular and biochemical properties have been well documented. Three Hevea A/N-Inv genes, namely HbNIN1, 2 and 3, were first cloned and characterized in planta and in Escherichia coli. Cellular localizations of HbNIN2 mRNA and protein were probed. From latex, active A/N-Inv proteins were purified, identified, and explored for enzymatic properties. HbNIN2 was identified as the major A/N-Inv gene functioning in latex based on its functionality in E. coli, its latex-predominant expression, the conspicuous localization of its mRNA and protein in the laticifers, and its expressional correlation with rubber yield. An active A/N-Inv protein was partially purified from latex, and determined as HbNIN2. The enhancement of HbNIN2 enzymatic activity by pyridoxal is peculiar to A/N-Invs in other plants. We conclude that HbNIN2, a cytosolic A/N-Inv, is responsible for sucrose catabolism in rubber laticifers. The results contribute to the studies of sucrose catabolism in plants as a whole and natural rubber synthesis in particular. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  3. Immunogenic cell death of human ovarian cancer cells induced by cytosolic poly(I:C) leads to myeloid cell maturation and activates NK cells.

    Science.gov (United States)

    Kübler, Kirsten; tho Pesch, Carola; Gehrke, Nadine; Riemann, Soheila; Dassler, Juliane; Coch, Christoph; Landsberg, Jennifer; Wimmenauer, Vera; Pölcher, Martin; Rudlowski, Christian; Tüting, Thomas; Kuhn, Walther; Hartmann, Gunther; Barchet, Winfried

    2011-10-01

    Owing to high rates of tumor relapse, ovarian cancer remains a fatal disease for which new therapeutic approaches are urgently needed. Accumulating evidence indicates that immune stimulation may delay or even prevent disease recurrence in ovarian cancer. In order to elicit proinflammatory signals that induce or amplify antitumor immune reactivity, we mimicked viral infection in ascites-derived ovarian cancer cells. By transfection or electroporation we targeted the synthetic double-stranded RNA poly(I:C) intracellularly in order to activate melanoma differentiation-associated gene-5 (MDA-5), a sensor of viral RNA in the cytosol of somatic cells. Cancer cells reacted with enhanced expression of HLA-class I, release of CXCL10, IL-6, and type I IFN as well as tumor cell apoptosis. Monocytes and monocyte-derived DCs (MoDCs) engulfed MDA-5-activated cancer cells, and subsequently upregulated HLA-class I/II and costimulatory molecules, and secreted CXCL10 and IFN-α. Further, this proinflammatory milieu promoted cytolytic activity and IFN-γ secretion of NK cells. Thus, our data suggest that the engagement of MDA-5 in a whole tumor cell vaccine is a promising approach for the immunotherapy of ovarian cancer. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. ZmHSP16.9, a cytosolic class I small heat shock protein in maize (Zea mays), confers heat tolerance in transgenic tobacco.

    Science.gov (United States)

    Sun, Liping; Liu, Yang; Kong, Xiangpei; Zhang, Dan; Pan, Jiaowen; Zhou, Yan; Wang, Li; Li, Dequan; Yang, Xinghong

    2012-08-01

    Various organisms produce HSPs in response to high temperature and other stresses. The function of heat shock proteins, including small heat shock protein (sHSP), in stress tolerance is not fully explored. To improve our understanding of sHSPs, we isolated ZmHSP16.9 from maize. Sequence alignments and phylogenetic analysis reveal this to be a cytosolic class I sHSP. ZmHSP16.9 expressed in root, leaf and stem tissues under 40 °C treatment, and was up-regulated by heat stress and exogenous H₂O₂. Overexpression of ZmHSP16.9 in transgenic tobacco conferred tolerance to heat and oxidative stresses by increased seed germination rate, root length, and antioxidant enzyme activities compared with WT plants. These results support the positive role of ZmHSP16.9 in response to heat stress in plant. The overexpression of ZmHSP16.9 enhanced tolerance to heat and oxidative stress in transgenic tobacco.

  5. Cytosolic phospholipase A2-alpha expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours.

    LENUS (Irish Health Repository)

    Caiazza, F

    2012-02-01

    BACKGROUND: The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)alpha expression correlated with EGFR\\/HER2 over-expression in a small number of breast cancer cell lines. METHODS: The importance of differential cPLA(2)alpha activity in clinical breast cancer was established by relating the expression of cPLA(2)alpha in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters. RESULTS: High cPLA(2)alpha mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA(2)alpha expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA(2)alpha expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up. CONCLUSION: This study shows a role of cPLA(2)alpha in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker.

  6. Presence of enolase in the M-band of skeletal muscle and possible indirect interaction with the cytosolic muscle isoform of creatine kinase.

    Science.gov (United States)

    Foucault, G; Vacher, M; Merkulova, T; Keller, A; Arrio-Dupont, M

    1999-01-01

    Glycerol-skinned skeletal muscle fibres retain the defined sarcomeric structure of the myofibrils. We show here that a small fraction of two enzymes important for energy metabolism, the cytosolic muscle isoform of creatine kinase (EC 2.7.3.2), MM-creatine kinase (MM-CK), and enolase (EC 4.2.1.11), remains bound to skinned fibres. CK is slowly exchangeable, whereas enolase is firmly bound. Two-dimensional gel electrophoresis followed by Western blot analyses demonstrates that both alpha (ubiquitous) and beta (muscle-specific) subunits of enolase are present in these preparations. Enolase and CK were co-localized at the M-band of the sarcomeres, as observed by indirect immunofluorescence and confocal microscopy. Cross-linking experiments were performed on skinned fibres with three bifunctional succinimidyl esters of different lengths and yielded a protein complex of 150 kDa that reacted with antibodies directed against either M-CK or beta-enolase. The cross-linking efficiency was greatest for the longest reagent and zero for the shortest one. The length of the cross-linker giving a covalent complex between the two enzymes does not support the notion of a direct interaction between M-CK and enolase. This is the first demonstration of the presence of an enzyme of energy metabolism other than CK at the M-band of myofibres. PMID:9931306

  7. Type I IFN and not TNF, is Essential for Cyclic Di-nucleotide-elicited CTL by a Cytosolic Cross-presentation Pathway

    Directory of Open Access Journals (Sweden)

    Darío Lirussi

    2017-08-01

    Full Text Available Cyclic di-nucleotides (CDN are potent stimulators of innate and adaptive immune responses. Cyclic di-AMP (CDA is a promising adjuvant that generates humoral and cellular immunity. The strong STING-dependent stimulation of type I IFN represents a key feature of CDA. However, recent studies suggested that this is dispensable for adjuvanticity. Here we demonstrate that stimulation of IFN-γ-secreting CD8+ cytotoxic T lymphocytes (CTL is significantly decreased after vaccination in the absence of type I IFN signaling. The biological significance of this CTL response was confirmed by the stimulation of MHC class I-restricted protection against influenza virus challenge. We show here that type I IFN (and not TNF-α is essential for CDA-mediated cross-presentation by a cathepsin independent, TAP and proteosome dependent cytosolic antigen processing pathway, which promotes effective cross-priming and further CTL induction. Our data clearly demonstrate that type I IFN signaling is critical for CDN-mediated cross-presentation.

  8. Over-expression of a cytosolic isoform of the HbCuZnSOD gene in Hevea brasiliensis changes its response to a water deficit.

    Science.gov (United States)

    Leclercq, J; Martin, F; Sanier, C; Clément-Vidal, A; Fabre, D; Oliver, G; Lardet, L; Ayar, A; Peyramard, M; Montoro, P

    2012-10-01

    Hevea brasiliensis is the main commercial source of natural rubber. Reactive oxygen species (ROS) scavenging systems are involved in various biotic and abiotic stresses. Genetic engineering was undertaken to study the strengthening of plant defences by antioxidants. To that end, Hevea transgenic plant lines over-expressing a Hevea brasiliensis cytosolic HbCuZnSOD gene were successfully established and regenerated. Over-expression of the HbCuZnSOD gene was not clearly related to an increase in SOD activity in plant leaves. The impact of HbCuZnSOD gene over-expression in somatic embryogenesis and in plant development are presented and discussed. The water deficit tolerance of two HbCuZnSOD over-expressing lines was evaluated. The physiological parameters of transgenic plantlets subjected to a water deficit suggested that plants from line TS4T8An displayed lower stomatal conductance and a higher proline content. Over-expression of the HbCuZnSOD gene and activation of all ROS-scavenging enzymes also suggested that protection against ROS was more efficient in the TS4T8An transgenic line.

  9. Blockage of cytosolic phospholipase A2 alpha sensitizes aggressive breast cancer to doxorubicin through suppressing ERK and mTOR kinases.

    Science.gov (United States)

    Li, Zhiqiang; Qu, Miao; Sun, Yang; Wan, Hongxing; Chai, Fang; Liu, Lihong; Zhang, Peng

    2018-01-29

    Advanced breast cancer is resistant to chemotherapy and its underlying mechanisms are not fully explored. In this work, we identified cytosolic phospholipase A2 alpha (cPLA2α) as a novel target to overcome chemoresistance in breast cancer. We demonstrated the increased transcriptional and translational expression of cPLA2α in breast cancer cells to acute and chronic exposure to doxorubicin. cPLA2α upregulation is also observed in breast cancer patients in response to chemotherapy. Inhibition of cPLA2α using two pharmacological inhibitors significantly enhances doxorubicin's effects to almost complete suppression in breast cancer cell growth, survival and migration. Similarly, depletion of cPLA2α significantly sensitizes breast cancer cells to doxorubicin treatment. We further found that cPLA2α inhibition led to decreased phosphorylation of ERK, mTOR, S6 and 4EBP1, suggesting the suppression of ERK and mTOR signaling pathways. These findings indicate the positive roles of cPLA2α in breast cancer cell growth, survival, migration and response to chemotherapy. Our work also highlights the therapeutic value of blocking cPLA2α to overcome chemoresistance in breast cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. In vitro Phase I and Phase II metabolism of α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV) and methedrone by human liver microsomes and human liver cytosol.

    Science.gov (United States)

    Negreira, Noelia; Erratico, Claudio; Kosjek, Tina; van Nuijs, Alexander L N; Heath, Ester; Neels, Hugo; Covaci, Adrian

    2015-07-01

    The aim of the present study was to identify the in vitro Phase I and Phase II metabolites of three new psychoactive substances: α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV), and methedrone, using human liver microsomes and human liver cytosol. Accurate-mass spectra of metabolites were obtained using liquid chromatography-quadrupole time-of-flight mass spectrometry. Six Phase I metabolites of α-PVP were identified, which were formed involving reduction, hydroxylation, and pyrrolidine ring opening reactions. The lactam compound was the major metabolite observed for α-PVP. Two glucuronidated metabolites of α-PVP, not reported in previous in vitro studies, were further identified. MDPV was transformed into 10 Phase I metabolites involving reduction, hydroxylation, and loss of the pyrrolidine ring. Also, six glucuronidated and two sulphated metabolites were detected. The major metabolite of MDPV was the catechol metabo